Mutant fragments of ospa and methods and uses relating thereto

ABSTRACT

The present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), a nucleic acid coding the same, a pharmaceutical composition (particularly for use as a medicament of in a method of treating or preventing a  Borrelia  infection) comprising the polypeptide and/or the nucleic acid, a method of treating or preventing a  Borrelia  infection and a method of immunizing a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/702,885, filed Dec. 4, 2019, which is a continuation of U.S.application Ser. No. 15/272,581, filed Sep. 22, 2016, granted as U.S.Pat. No. 10,544,194, which is a continuation of U.S. application Ser.No. 14/412,722, filed Jan. 5, 2015, which is a national stage filingunder 35 U.S.C. § 371 of international application PCT/EP2013/064403,filed Jul. 8, 2013, which was published under PCT Article 21(2) inEnglish, claims the benefit under 35 U.S.C. § 120 of U.S. applicationSer. No. 13/802,991, filed Mar. 14, 2013, granted as U.S. Pat. No.8,986,704, and claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 61/668,627, filed Jul. 6, 2012, thedisclosures of which are incorporated by reference herein in theirentireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(I042270092US10-SUBSEQ-NTJ.xml; Size: 381,637 bytes; and Date ofCreation: Apr. 14, 2023) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of Borrelia infection. Particularly, thepresent invention relates to a polypeptide comprising a mutant fragmentof an outer surface protein A (OspA), a nucleic acid coding the same, apharmaceutical composition (particularly for use as a medicament of in amethod of treating or preventing a Borrelia infection) comprising thepolypeptide and/or the nucleic acid, a method of treating or preventinga Borrelia infection and a method of immunizing a subject.

BACKGROUND OF THE INVENTION

Lyme borreliosis, or Lyme disease, is the most commonly reportedtick-borne disease in Europe and North America. The disease is caused bythe arthropod-borne gram-negative-like spirochete. Borrelia burgdorferisensu lato (B. burgdorferi s.l.), and is an infection that can involvemultiple organs or tissues, resulting in skin, cardiac, musculoskeletaland neurological disorders. In most countries, Lyme borreliosis is not anotifiable disease and no exact data regarding annual incident rates areavailable. In the United States, the causative agent is B. burgdorferisensu stricto (B. burgdorferi s.s.) and Lyme borreliosis is localized tonorth-eastern, mid-Atlantic and upper north-central states. In 2010, atotal of about 30,000 cases of Lyme borreliosis were reported for the USto the Centers for Dirac& Control and Prevention (CDC). In Europe, B.afzelii and B. garinii are the main causative agents of Lymeborreliosis, as well as B. burgdorferi s.s, and B. bavariensis, whichcontribute to a lesser extent depending on the geographic location. Theprevalence of Lyme borreliosis varies considerably in different Europeancountries with an overall increased prevalence from west to east. Inmuch of Europe, the number of reported cases of Lyme borreliosis hasincreased since the early 1990s (e.g., the Czech Republic, Estonia.Lithuania: see Lyme borreliosis in Europe. WHO report of 2006), and thegeographic distribution of cases has also expanded. Borrelia belongs tothe family Spirochaetaceae, which is subdivided into the medicallyimportant genera Treponema, Leptospira and Borrelia. B. burgdorferi s.l.is a spiral-shaped, vigorously motile gram-negative bacterium, about10-20 μm long and 0.2-0.5 μm wide, that grows under microaerophilicconditions. The spirochetal cell wall consists of a cytoplasmic membranesurrounded by peptidoglycan and several flagella and then by aloosely-associated outer membrane.

Lyme borreliosis generally occurs in stages characterized by differentclinical manifestations, with remissions and exacerbations. Stage 1,early infection consists of a localized infection of the skin.

followed within days or weeks by stage 2, disseminated infection, andmonths to years later by stage 3, persistent infection. However, theinfection is variable; some patients have only localized infections ofthe skin, while others display only later manifestations of the illness,such as arthritis. Different clinical syndromes of Lyme borreliosis arealso caused by infection with diverse B. burgdorferi s.l. species. B.burgdorferi s.s, more often causes joint manifestations (arthritis) andheart problems, B. afzelii causes mainly dermal symptoms (erythemamigrans; EM and acrodermatitis chronica atrophicans; ACA), whereas B.garinii is implicated in most cases of neuroborreliosis.

Localized infection—The most common symptom of stage 1 of an infectionis erythema migrans, which occurs in 70-80% of infected people. Thisskin lesion is often followed by flu-like symptoms, such as myalgia,arthralgia, headache and fever. These non-specific symptoms occur in 50%of patients with erythema migrans.

Disseminated infection—During stage 2, the bacteria move into the bloodstream from the site of infection to distal tissues and organs.Neurological, cardiovascular and arthritic symptoms that occur in thisstage include meningitis, cranial neuropathy and intermittentinflammatory arthritis.

Persistent infection—Stage 3 of the infection is chronic and occurs frommonths to years after the tick bite. The most common symptom in NorthAmerica is rheumatoid arthritis, caused by an infection with B.burgdorferi s.s. Persistent infection of the central nervous system withB. garinii causes more severe neurological symptoms during stage 3, anda persistent infection of the skin with B. afzelii results inacrodermatitis chronica atrophicans.

In some risk groups, such as farmers, forestry workers, hikers, runnersor vacationers, seroprevalence and disease incidence rates haveincreased, as in children under 15 years of age and adults between 39and 59, without gender preference. This increased incidence of Lymeborreliosis is linked to changes in forest habitats as well as socialfactors. Environmental changes, such as forest fragmentation, have ledto a sharp reduction of rodent predators such as foxes and birds ofprey, which in turn has led to an increase in the mouse population, witha subsequent increase in the tick population. More recently, patchyreforestation has increased the number of deer and thus the number ofticks. Suburban sprawl and the increasing use of woodland areas forrecreation such as camping and hiking has brought humans into greatercontact with the larger number of tick Borrelia vectors. All of thesefactors together have contributed to a wider distribution of Borreliaand a higher incidence of Lyme borreliosis.

Antimicrobial agents are the principle method of treatment of Borreliainfection. The antibiotic used depends on the stage of the disease,symptoms, and the patient's allergies to medication. The length of theantibiotic course also depends on the stage of the disease and theseverity of symptoms. Early Lyme borreliosis is typically treated withoral tetracyclines, such as doxycycline, and semi-synthetic penicillin,such as amoxicillin or penicillin V. Arthritic and neurologicaldisorders are treated with high-dose intravenous penicillin G orceftriaxone. Up to 30% of Lyme borreliosis patients do not display theearly characteristic symptoms of infection with Borrelia, makingdiagnosis and treatment problematic. The antibiotic course can be long(up to several months) and sometimes ineffective and is thus debated inthe Borrelia field, especially during later-stage disease. Even in thecase of effective treatment of Borrelia, patients can be left withdebilitating fatigue, pain, or neurological symptoms for yearsafterwards referred to as post-treatment Lyme disease syndrome. Ingeneral, the use of antibiotics can have undesirable consequences, suchas the development of resistance by the target micro-organisms. Finally,antibiotic therapy may effectively cure Lyme borreliosis, but providesno protection against subsequent infections.

A monovalent serotype 1-OspA-based vaccine (LYMErix™) was approved andmarketed in the USA for the prevention of Lyme disease caused byBorrelia burgdorferi s.s. However, heterogeneity in OspA sequencesacross different serotypes in Europe and elsewhere precludes efficientprotection with a vaccine based on OspA from only a single serotype.

Chimeric OspA molecules comprising the proximal portion from one OspAserotype, together with the distal portion form another OspA serotype,while retaining antigenic properties of both of the parent polypeptides,may be used in the prevention and treatment of Lyme disease orborreliosis (WO2011/143617, WO2011/143623).

Currently, there is no preventative medicament for Lyme borreliosis onthe market and thus there is a need in the art for the development ofsuch a medicament that can provide effective protection against Borreliathat are present in the USA, Europe and elsewhere, especially for thedevelopment of a medicament that can provide effective protectionagainst several Borrelia serotypes simultaneously.

SUMMARY OF THE INVENTION

The present invention relates to a polypeptide comprising a mutantfragment of Borrelia outer surface protein A (OspA), a nucleic acidencoding the same, a vector which comprises such nucleic acid molecule,and a host cell comprising such vector. Furthermore, the inventionprovides a process for producing such polypeptide and a process forproducing a cell which expresses such polypeptide. Moreover, the presentinvention provides antibodies specifically binding to such polypeptide,a hybridoma cell producing such antibodies, methods for producing suchantibodies, a pharmaceutical composition comprising such polypeptide,nucleic acid molecule, vector or antibody, the use of such polypeptide,nucleic acid molecule, vector or antibody for the preparation of amedicament or a pharmaceutical composition (particularly for use as avaccine or in a method of treating or preventing a Borrelia infection),methods for diagnosing an infection and methods for treating orpreventing a Borrelia infection and methods of immunizing a subject.

Efforts to develop a subunit vaccine for prevention of Lyme borreliosishave been focused in large part on the use of borrelial outer surfaceprotein A (OspA) as an antigen. The OspA protein is expressed byBorrelia only when it is in the gut of the tick vector. Thus, OspAantibodies produced by vaccination do not fight infection in the body,but rather enter the gut of the tick when it takes a blood meal. There,the antibodies neutralise the spirochetes and block the migration ofbacteria from the midgut to the salivary glands of the tick, the routethrough which Borrelia enters the vertebrate host. Thus, OspA-specificantibodies prevent the transmission of Borrelia from the tick vector tothe human host.

The lipidated form of OspA from B. burgdorferi s.s., strain ZS7,together with aluminium hydroxide was commercially developed as avaccine against Borrelia (LYMErix™) by SmithKline Beecham, nowGlaxoSmithKline (GSK) for the US market. Three doses of LYMErix® over aperiod of one year were needed for optimal protection. After the firsttwo doses, vaccine efficacy against Lyme borreliosis was 49%, and afterthe third dose 76%. However, shortly after LYMErix™ was commerciallyavailable, it was withdrawn from the market in 2002. Reasons cited werematters of practical application of the vaccine, for example the needfor booster injections every year or every other year, as well as therelatively high cost of this preventive approach compared withantibiotic treatment of early infection. In addition, there was aconcern that LYMErix™ could trigger autoimmune reactions in a subgroupof the population due to sequence homology with a human protein, thoughthis was never proven. In addition, cross-protection against otherclinically important Borrelia species was not provided by this vaccine.

Accordingly, in one embodiment, it was an object of the presentinvention to provide an improved vaccine for the prevention of Lymeborreliosis. Preferably, the vaccine is easily produced while beingprotective, safe and more effective than existing therapies and/orprovides protection against more than one Borrelia species.

The problem underlying the present invention is solved by a polypeptidecomprising a mutant fragment of an outer surface protein A (OspA),wherein the mutant fragment consists of a C-terminal domain of an OspAprotein of Borrelia and differs from the corresponding wild-typefragment at least by the introduction of at least one disulfide bond.

Surprisingly, it was found that the introduction of at least onedisulfide bond in a mutant fragment increases the protective capacity ofthe polypeptide comprising the mutant OspA fragment relative to apolypeptide comprising the wild-type OspA fragment, as shown in an invivo model of infection. As shown in the Examples, the introduction ofat least one disulfide bond into the B. afzelii OspA C-terminal fragmentincreased its protective capacity relative to the wild-type OspAfragment without a disulfide bond. Tables 2 and 3 provide datademonstrating the protective capacity of mutant fragments with anintroduced disulfide bond (“S2D1-5”) as compared to the wild-type OspAfragment (“S2D0”), as fewer animals were infected after immunizationwith mutant OspA fragments in comparison to wild-type OspA fragments.Some of the mutant OspA fragments tested provided protection comparableto that conveyed by the positive control antigen, the non-lipidatedfull-length OspA protein.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect, the present invention relates to apolypeptide comprising a mutant fragment of an outer surface protein A(OspA), wherein the mutant fragment consists of a C-terminal domain ofan OspA of Borrelia and differs from the corresponding wild-typefragment at least by the introduction of at least one disulfide bond.

The term B. burgdorferi s.l. encompasses at least 13 Borrelia species(Table A-1). These species occur in different geographic regions, andlive in nature in enzootic cycles involving ticks of the Ixodes ricinuscomplex (also called Ixodes persulcatus complex) and a wide range ofanimal hosts. Four Borrelia species are responsible for the majority ofinfections in humans: B. burgdorferi s.s., B. afzelii, B. bavariensisand B. garinii. Three other species, B. lusitaniae, B. bissettii and B.spielmanii, have occasionally been detected in humans, but their role inLyme borreliosis is uncertain at present. New species of Borrelia arestill being reported.

TABLE A-1 Principal tick vector Location Pathogenic species (4) Borreliaburgdorferi Ixodes scapularis Northeastern/ (Borrelia burgdorferi s.s.)Ixodes pacificus north-central US Ixodes ricinus Western US Ixodespersulcatus Europe Asia Borrelia garinii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia afzelii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia bavariensis Ixodes ricinus Europe Ixodespersulcatus Asia Minimally pathogenic or non-pathogenic species (9)Borrelia andersonii Ixodes dentatus Eastern US Borrelia bissettii Ixodesspinipalpis Western US Ixodes pacificus Europe Ixodes ricinus Borreliavalaisiana Ixodes ricinus Europe and Ixodes columnae Asia Borrelialusitaniae Ixodes ricinus Europe Borrelia spielmanii Ixodes ricinusEurope Borrelia japonica Ixodes ovatus Japan Borrelia tanukii Ixodestanuki Japan Borrelia turdi Ixodes turdus Japan Borrelia sinica Ixodespersulcatus China

As detailed above, Borrelia outer surface protein A (OspA) is anabundant immunogenic lipoprotein of Borrelia of particular interestbecause of its potential as a vaccine candidate. OspA of B. burgdorferis.l. is a basic lipoprotein that has a molecular mass of approximately30 kDa and is encoded on a linear plasmid. An important aspect of theOspA protein is its N-terminal lipidation; that is, the N-terminalcysteine residue is substituted with fatty acids with a chain length ofbetween C14 and C19 with or without double-bonds, a feature thatenhances the immunogenicity of the OspA protein. It has been shown thatpoorly-immunogenic synthetic peptides induce stronger antibody responseswhen lipidated: for example, when covalently coupled to Pam₃Cys (Besslerand Jung, Research Immunology (1992) 143:548-552), a fatty acidsubstitution found at the amino terminus of many bacterial lipoproteinsthat are synthesized with a signal sequence specifying lipid attachment.Additionally, the Pam₃Cys moiety was shown to enhance immune responsesto OspA in mice, partially through its interaction with TLR-2 (Yoder, etal. (2003) Infection and Immunity 71:3894-3900). Therefore, lipidationof a C-terminal fragment of OspA would be expected to enhance theimmunogenicity and protective capacity of the fragment.

Analysis of isolates of B. burgdorferi s.l, obtained in North Americaand Europe has revealed that OspA has antigenic variability and thatseveral distinct groups can be defined based on serology. Anti-OspA mAbswhich bind to specific N- and C-terminal antigenic determinants havebeen reported. X-ray crystallography and NMR analysis have been used toidentify immunologically important hypervariable domains in OspA andhave mapped the LA-2 epitope to C-terminal amino acids 203-257 (Ding etal., Mol. Biol. 302: 1153-64, 2000). Previous studies have shown thatthe production of antibodies against the C-terminal epitope LA-2correlates with protective immunity after vaccination with OspA (VanHoecke et al. Vaccine (1996) 14(17-18):1620-6 and Steere et at, N Engl JMed (1998) 339:209-215). Antibodies to LA-2 were shown to block thetransmission of Borrelia from tick to host (Golde et al., Infect Immun(1997) 65(3):882-889). These studies suggested that the C-terminalportion of the OspA protein may be sufficient for inducing protectiveimmunity. It should be noted that the sequence of the C-terminal portionof OspA is less highly-conserved between Borrelia serotypes than is theN-terminal portion (see FIG. 1 ).

Based on information from the studies outlined above, along with others,truncated forms of OspA comprising the C-terminal portion (also referredto herein as “OspA fragment” or “monomer”) were used in the currentinvention. These truncated forms of OspA proved to be less protectivethan the full-length OspA protein. Surprisingly, however, it was foundin the course of the current invention that the introduction of adisulfide bond in the truncated form (also referred to herein as “mutantOspA fragment” or “mutant fragment”) overcomes this disadvantage. Whilenot being limited to a specific mechanism, it is thought that improvedprotection is due to increased stability of the OspA fragment, as shownin assays measuring thermal stability.

In accordance with the present invention, the mutant OspA fragment maybe derived from any Borrelia species; however, due to their relevance inthe medical field, particularly for humans, B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii are preferred. In this regard,these four Borrelia species can be further classified according to theirOspA serotypes, which have been determined by analysis with monoclonalantibodies specific to the respective OspA protein. Serotypes 1-7, whichaccount for the majority of human Borrelia infections, along with theirrates of prevalence, are shown in Table A-2 below.

TABLE A-2 Serotype designation and prevalence of B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii. Borrelia isolated from humancerebrospinal fluid or skin or from tick vectors were serotyped byprobing whole-cell lysates with mouse monoclonal antibodies, eachspecific to a particular epitope of OspA (as described by Wilske et al.,J. of Clin Microbiol (1993) 31(2): 340-350 and presented by BaxterBioscience at “Climate change effect on ticks and tick-borne diseases”,Brussels, 6 Feb. 2009). OspA serotype Prevalence Strain defined by mAbin human source for Seq Borrelia sp. testing disease sequence ID No: B.burgdorferi s.s. 1 11% B31 20 B. afzelii 2 63% K78 19 B. garinii 3 1.5% PBr 21 B. bavariensis 4  4% PBi 22 B. garinii 5  6% PHEi 23 B. garinii 613% DK29 24 B. garinii 7 0.5%  T25 25

The structure of the OspA protein from B. burgdorferi s.s. strain B31was determined by Li et al. (Proc Natl Acad Sci (1997) 94:3584-3589). Itis composed of N-terminal (β-strands 1 to 4) and central β-sheets(β-strands 5 to 14n [N-terminal part]), barrel sheet 1 (β-strands 14c[C-terminal part] to 16), barrel sheet 2 (β-strands 17 to 21) and aC-terminal α-helix. The term “OspA C-terminal domain” or “C-terminaldomain” or “wild-type fragment” or “C-terminal portion” with respect toOspA as used throughout the present specification shall mean theC-terminal amino acid sequence of OspA, i.e., OspA lacking at least theN-terminal β-sheet (including 0-strands 1 to 4). In OspA from B.burgdorferi s.s. strain B31, the N-terminal sheet consists of aminoacids 17 to 70 (following post-translational cleavage of the 16 as longlipidation signal peptide).

The C-terminal OspA fragment of the current invention may also include alipidation signal sequence at the N-terminus, e.g., the lipidationsignal sequence of amino acids t to 16 of OspA (SEQ ID NO: 14) or OspB(SEQ ID NO: 15) from B. burgdorferi s.s. strain B31, a lipidation signalsequence from E. coli, referred to herein as the “lpp lipidation signal”(SEQ ID NO: 16), or any other signal sequence, e.g., as defined below.

Lipidation of a protein with an N-terminal lipidation signal sequence,such as those present on a nascent OspA polypeptide, occurs in the E.coli expression vector by the step-wise action of the enzymesdiacylglyceryl transferase, signal peptidase II and transacylase,respectively. The first step is the transfer of a diacylglyceride to thecysteine sulphydryl group of the unmodified prolipoprotein, followed bythe cleavage of the signal peptide by signal peptidase II and, finally,the acylation of the α-amino group of the N-terminal cysteine of theapolipoprotein. The result is the placement of one lipid and a glycerolgroup substituted with two further lipids on the N-terminal cysteineresidue of the polypeptide. The lipidation signal sequence, which iscleaved off during lipidation, is not present in the final polypeptidesequence.

According to the current invention, the mutant OspA fragment may be alipidated protein, also lipoprotein, wherein the lipid moieties, alongwith the glycerol group, is also referred to as “Lip”. According to theinvention, Lip comprises one to three lipids such as C₁₄₋₂₀ alkyl and/orC₁₄₋₂₀ alkenyl attached to a glycerol and the N-terminal cysteine of thepolypeptide of the invention, or preferably wherein Lip is a moiety offormula (I) below,

in which one of R₁, R₂ or R₃ is C₁₄-C₂₀ alkyl or alkenyl, and each ofthe others, independently is C₁₄-C₂₀ alkyl or C₁₄-C₂₀ alkenyl, and X isan amino acid sequence attached to the cysteine residue shown in Formula(I). More preferably, Lip plus the N-terminal cysteine of thepolypeptide is N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propylcysteine (referred to herein as “Pam₃Cys”) and is connected via thecarbonyl C of the cysteine to said amino acid sequence of the invention.In Formula (I) above R₁, R₂ and R₃ would be palmitoyl moieties and X isan amino acid sequence attached to the cysteine residue.

In accordance with the current invention, the C-terminal domain of anOspA from a strain other than B. burgdorferi s.s. B31 is defined by (i)lacking at least amino acids 17 to 70 and/or (ii) by lacking at leastthe N-terminal domain homologous to amino acids 17 to 70 of OspA from B.burgdorferi s.s. B31. Additionally, the OspA C-terminal domain accordingto the present invention may also lack further portions of the centralsheet as defined by Li and co-workers (Li el al., supra), particularlyfurther strands such as the amino acid portions from amino acid 17 to82, 93, 105, 118 or 119, preferably 17 to 129, more preferably 1 to125.1 to 129 or 1 to 130 of any Borrelia, particularly B. burgdorferis.s. B31, or homologous portions of an OspA protein from a Borrelia sp.other than B. burgdorferi s.s. B31.

In the context of the present invention, the OspA C-terminal domain isalso referred to as “OspA fragment” or “fragment of OspA”.

The “mutant fragment” in the context of the polypeptide of the presentinvention and as used throughout the present specification shall meanthe OspA C-terminal fragment, as defined above, which differs from thewild-type fragment at least by at least two introduced cysteines thatcan form a disulfide bond. Without being bound to that theory, it isassumed that the disulfide bond stabilizes the fragment in aconformation conducive to the induction of antibody binding. The fold ofthe wild-type C-terminal fragment of OspA shows reduced temperaturestability in comparison to the full-length protein (Koide et al.,Structure-based Design of a Second-generation Lyme Disease Vaccine Basedon a C-terminal Fragment of Borrelia burgdorferi OspA, J. Mol. Biol.(2005) 350:290-299). For the present invention, the sequence of theC-terminal domain of the B. burgdorferi s.s. B31 OspA has been in silicoanalyzed to determine positions for introduced disulfide bridges thatmay enhance the stability of the fold of this C-terminal domain. Theresults of the analysis have been transferred to homologous OspAfragments of other Borrelia species with the assumption that the fold isconserved across species.

Typically, the disulfide bond may be introduced by the introduction ofone or more cysteine residues, wherein a disulfide bond (S—S bridge) isformed between the thiol groups of two cysteine residues. Only onecysteine residue need be introduced if a disulfide bond is formed with acysteine residue present in the wild-type fragment. The one, orpreferably two, cysteine(s) may be introduced by amino acid addition or,preferably, substitution.

The OspA mutant fragment may also comprise further mutations relative tothe wild-type. As detailed above, the structure and surface domain ofOspA are known in the art. Accordingly, the mutant fragment may comprisefurther mutations, particularly at sites not on the surface of theprotein and/or nut involved in the immune response and, therefore notimpacting antigenic capacity. These can include one or more amino aciddeletion(s), particularly small (e.g., up to 10 amino acids) deletions,one or more amino acid addition(s) (particularly C- or N-terminally),one or more amino acid substitution(s), particularly one or moreconservative amino acid substitutions. Examples of conservative aminoacid substitutions include, but are not limited to, those listed below:

Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp His Asn;Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Asn Met Leu; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Preferred mutations include changes in selected portions of thefragment, for example, wherein the sequence with sequence similarity tohuman leukocyte function-associated antigen (hLFA-1), which exists in B.burgdorferi s.s., is modified, for example, replaced by a homologoussequence from an OspA protein from another Borrelia sp. The rationalefor this modification is to reduce the risk for inducing immunologicalcross-reaction with human proteins. Also possible is the addition of asignal sequence for lipidation in the final, or an intermediate,fragment, or the addition of a marker protein (e.g., for identificationor purification).

In some embodiments, the mutant OspA fragment has an amino acid sequencethat has 60%, preferably at least 70%, more preferably at least 80%,more preferably 85%, more preferably 90%, even more preferably 95%sequence identity to the wild-type fragment. In another embodiment, thesequence differs by at most 10%, at most 9%, at most 8%, at most 7%, atmost 6%, 5%, 4%, 3%, 2%, most preferably at most 1%, due to a sequenceaddition, deletion or substitution.

Identity, as known in the art and as used herein, is the relationshipbetween two or more polypeptide sequences, as determined by comparingthe sequences. In the art, identity also means the degree of sequencerelatedness between polypeptide or polynucleotide sequences, as the casemay be, as determined by the match between strings of such sequences.Identity can be readily calculated. While a number of methods exist tomeasure identity between two polynucleotides or two polypeptidesequences, the term is well known to skilled artisans (e.g. Sequenceanalysis in Molecular Biology, von Heinje, G., Academic Press, 1987).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity arecodified in computer programs. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN,and FASTA (Altschul, S. et al., 1990).

In contrast to the mutant OspA fragment, the “wild-type fragment” in thecontext of the present invention relates to a fragment of anaturally-occurring OspA of Borrelia. The wild-type fragment is obtainedby N-terminal deletions, but it does not comprise internal deletions(except from signal sequences as detailed herein) or mutations. Inrelation to the mutant OspA fragment, the wild-type fragment consists ofan identical part of the OspA (identical length and same strain of OspA,etc.) and differs only in the mutation(s) detailed above, particularlyin the introduction of at least one disulfide bond or the replacement ofa sequence with human homology, for example hLFA-1 (see above).

According to a preferred embodiment of the present invention, thepolypeptide of the present invention does not comprise or consist of thefull-length OspA polypeptide having at least one disulfide bondintroduced.

In one embodiment of the present invention, the mutant OspA fragment maydiffer from the respective wild-type fragment only by the introductionof at least one, preferably exactly one, disulfide bond.

A polypeptide is a single linear polymer of amino acids linked bypeptide bonds, in some cases also by disulfide bonds. In accordance withthe present invention, the polypeptide may also compromise one or moreposttranslational modifications: i.e., an attached biochemicalfunctional group, such as an attached acetate, phosphate, lipid orcarbohydrate, preferably a lipid or lipids attached to the N-terminalcysteine along with a glycerol, more preferably 1 to 3 C₁₄-C₂₀ alkyl oralkenyl moieties, even more preferably 1 to 3 palmitoyl groups, mostpreferably three palmitoyl groups (Pam).

In accordance with the present invention, the polypeptide of the presentinvention comprises the above-described mutant OspA fragment. Accordingto the present invention, it does not comprise (i) the N-terminal sheetas defined above and (ii) optionally one or more further strands of thecentral sheet as defined above. However, the polypeptide may compriseone or more functional sequences such as a signal sequence, e.g., alipidation signal sequence or a posttranslational modification, such aslipidation.

In a further embodiment of the present invention, the polypeptide of thepresent invention consists of (i) one or more mutant OspA fragments,optionally joined by linkers, e.g., as defined below and (ii) optionallyone or more amino acids heterologous to OspA, particularly a signalsequence and (iii) optionally a posttranslational modification, such aslipidation.

The polypeptide of the present invention has protective capacity. Asdetailed above, the introduction of a disulfide bond into the mutantOspA fragment increases the protective capacity of the polypeptiderelative to a polypeptide comprising the respective fragment without thedisulfide bond(s). In some embodiments, the protective capacity isincreased by at least 10%, more preferably by at least 20%, morepreferably by at least 30%, more preferably by at least 40%, morepreferably by at least 50%, more preferably by at least 60%, morepreferably by at least 70%, more preferably by at least 80%, even morepreferably by at least 90% relative to a polypeptide comprising therespective fragment without the disulfide bond(s).

The term protective capacity describes the ability to protect a subjectagainst a Borrelia infection. With respect to the polypeptide of theinvention, protective capacity relates to the ability of the polypeptideto induce an immune response that protects a subject against a Borreliainfection. Protective capacity can be tested by administering to asubject the polypeptide in a manner to induce an immune reaction againstthe polypeptide. Thereafter, the subject may be challenged withBorrelia. The subject's reaction to the infection is monitored.Particularly, the presence of Borrelia in the subject may be determined.For example, the polypeptide is protective if Borrelia cannot bedetected in the subject. The presence of Borrelia can be determined bydetecting Borrelia-specific nucleic acids (e.g., by PCR) orBorrelia-specific antibodies (e.g., by ELISA or Western blot) or bydetecting Borrelia itself (e.g., culturing organs or tissues in growthmedium and verifying the presence of Borrelia by microscopy). Inparticular, the protective capacity (“pc”), reported as a percentage,for a particular dose is defined as follows:

pc (%)=[(number of total tested subjects−number of Borrelia-infectedsubjects)/number of total tested subjects]×100

Differences in protective capacity (Δpc) may be determined by, e.g.comparing the protective capacity (pc) of a mutant OspA fragment with adisulfide bond(s) (pc [with bond]) to the protective capacity of an OspAfragment without a disulfide bond(s) (pc [w/o bond]). In accordance withthe present invention, the polypeptides to be compared differ only inthe introduction of at least one disulfide bond. The change inprotective capacity (Δpc) by the introduction of the disulfide bonds) isdetermined as follows:

Δpc=(pc[sample]−pc[control])

e.g. Δpc=(pc[with bond]−pc[w/o bond])

If Δpc is greater than zero (>0), assuming all other parameters (e.g.,dose and assay) are the same, then the protective capacity of the sample(e.g. the mutant OspA fragment with a disulfide bond(s)) is better thanthe protective capacity of the control (e.g. the OspA fragment without adisulfide bond(s)). Conversely, if Δpc is less than zero (<0) andassuming all other parameters (e.g., dose and assay) are the same, thenthe protective capacity of the sample (e.g. the mutant OspA fragmentwith a disulfide bond(s)) is less than the protective capacity of thecomparison (e.g., the OspA fragment without a disulfide bond(s)).

Preferably, the polypeptide of the present invention is assessed for itsprotective capacity by an in vivo challenge assay wherein mice immunizedwith the polypeptide of the invention or with a placebo control arechallenged with Borrelia introduced into the immunized subjects with ahypodermic needle (Needle Challenge Method) or by introduction by a tickvector (Tick Challenge Method).

The Needle Challenge Method is carried out for the desired Borreliastrain (e.g., B. burgdorferi, strain N40) by subcutaneously introducingBorrelia at a dose between 20 and 50 times the Infectious Dose (ID)₅₀ tomice that are immunized with said first polypeptide of the first aspector with an appropriate placebo (negative) control, such as buffer oradjuvant alone and comparing the rates of infection in the challengedmice. The ID₅₀ is defined as the dose at which 50% of the challengedmice are infected. The dose of Borrelia is measured in numbers ofbacteria. The challenge dose can vary widely and is strain-dependent;therefore, the virulence of the strain must first be assessed bychallenge experiments for determination of ID₅₀. Four weeks after needlechallenge, blood and tissues are collected for readout methods todetermine the infection status. These readout methods can be e.g. VIsEELISA on sera or qPCR on collected tissues for identification ofBorrelia, as described herein, or other methods.

The Tick Challenge Method is carried out by applying at least one ticknymph (e.g., I. ricinus) infected with Borrelia (e.g., B. afzelii,strain IS1), to a mouse that is immunized with said first polypeptide ofthe first aspect; and b) applying at least one infected tick nymph to asecond mouse that is immunized with said second polypeptide of the firstaspect; and c) comparing the rates of infection in the two mice,generally six weeks after challenge. Preferably, the assay or test isdone with a group of mice per polypeptide to be tested. A suitable testis also described and illustrated in the Examples. Assessment ofinfection status can be done using VIsE ELISA on sera or qPCR oncollected tissues, or using other suitable methods.

In a preferred embodiment of the present invention, the products of theinvention such as, e.g. the polypeptides of the invention comprising themutant OspA fragment with a disulfide bond(s) administered 3 times to asubject at a dose of 30 μg, preferably 10 μg, preferably 5.0 μg,preferably 1.0 μg, preferably 0.3 μg or lower have a protective capacityof 50% or more, preferably 60% or more, more preferably 70% or more,more preferably 80% or more, more preferably 90% or more, even morepreferably 95% or more, most preferred 99% or more. In one embodiment,the protective capacity is assessed in an in vivo challenge method,preferably a Tick Challenge Method, more preferably a Needle ChallengeMethod, e.g. as described in the Examples. It has been surprisinglyobserved that immunization with an OspA mutant fragment of one serotypecan provide cross-protection against other another serotype (Example 4,Table 4). Based on this finding, it might be anticipated that the doseof polypeptide of the present invention could be even further reduced.

In a preferred embodiment, the difference in protective capacity (Δpc)between the polypeptides of the invention comprising the mutant OspAfragment with a disulfide bond(s) and the placebo (negative) control isat least 50%, especially at least 60%, preferably at least 70%, morepreferably at least 80%, even more preferably 90%, even more preferably95%, most preferably at least 95%, when administered 3 times to asubject at a dose of 30 μg, preferably 10 μg, preferably 5.0 μg,preferably 1.0 μg, preferably 0.3 μg or lower.

In a preferred embodiment of the present invention, the C-terminaldomain is defined as a fragment consisting of at least the C-terminal150 amino acids of the OspA protein. In one embodiment, the C-terminaldomain is between 140 and 152 amino acids in length. In a furtherembodiment, the C-terminal domain consists of no more than the last 152amino acids of the OspA protein, preferably the last 151 amino acids,more preferably the last 150 amino acids. In an alternative embodiment,the C-terminal domain consists of no less than the last 140 amino acidsof the OspA protein, preferably the last 141 amino acids, preferably thelast 142 amino acids, most preferably the last 143 amino acids. The lastamino acids of the OspA protein are defined herein as the mostC-terminal contiguous amino acid sequence of the OspA protein.

In another embodiment, the C-terminal domain of an OspA protein ofBorrelia comprises, essentially consists of or consists of (i) the aminoacids from position 126, 131 or 130 to position 273 of the OspA of B.afzelii, strain K78 or (ii) the homologous domain to amino acids of OspAfrom a Borrelia strain other than B. afzelii, strain K78.

The polypeptide of the present invention may comprise or essentiallyconsists of or consist of (i) one or more of these mutant fragments,optionally joined by linkers, e.g., as defined below and (ii) optionallyone or more amino acids heterologous to OspA, particularly a signalsequence or site for a post-translational modification such aslipidation and (iii) optionally a posttranslational modification, suchas lipidation.

In accordance with the present invention, the polypeptide of the presentinvention may comprise or essentially consists of or consist of of theelements as defined herein, particularly the one or more mutant OspAfragments and optionally one or more further elements such as homologousdomain, a linker peptide, a signal sequence or a site for lipidation.“Essentially consists” in this context means that the element(s) mayhave some minor amino acid changes with respect to the above sequences,such as amino acid additions, substitutions or deletions, preferablyrelating to at most 10%, 5%, 4%, 3%, 2% or 1% of the amino acids of theelements as defined herein.

In accordance with the present invention, at least one disulfide bond isintroduced into an OspA fragment. This may preferably be achieved byintroducing into the fragment at least 1 or 2 cysteine(s), particularly2 cysteines, in order to allow for the formation of the at least onedisulfide bond. Only one cysteine may be introduced, if another cysteinein the fragment is available for a disulfide bond. However, preferablytwo cysteines are introduced. The cysteine(s) is/are introduced by aminoacid addition or substitution, preferably substitution. In case ofaddition, the cysteine is inserted into the amino acid sequence betweentwo amino acids, whereas in case of substitution one amino acid isreplaced with the cysteine.

In accordance with the present invention, the OspA may be from anyBorrelia strain, particularly from those specified herein such as B.burgdorferi s.s., B. garinii, B. afzelii, B. andersoni, B. bissettii, B.valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B.turdi or B. sinica, B. bavariensis, preferably from B. burgdorferi s.s.,B. afzelii, B. bavariensis or B. garinii. Preferably, the OspA is fromB. afzelii, particularly strain K78, OspA serotype 2 (SEQ ID NO: 19); B.burgdorferi s.s., particularly strain B31, OspA serotype 1 (SEQ ID NO:20); B. garinii, particularly strain PBr, OspA serotype 3 (SEQ ID NO:21); B. bavariensis, particularly strain PBi, OspA serotype 4 (SEQ IDNO: 22); B. garinii, particularly strain PHei, OspA serotype 5 (SEQ IDNO: 23); B. garinii, particularly strain DK29, OspA serotype 6 (SEQ IDNO: 24) or B. garinii, particularly strain T25, OspA serotype 7 (SEQ IDNO: 25). The amino acid sequences of these OspA proteins (full-length)are given below.

TABLE A-3 Accession numbers of OspA sequences from selected strains ofBorrelia species. Organism_Strain db|accession.version Organism_Straindb|accession.version Organism_Strain db|accession.version Bbu_156a(serotype 1) gb|ACL33776.1 Bbu_K48 emb|CAA44492.1 Bga_Mng4702gb|ABF29559.1 Baf_K78 (serotype 2) emb|CAA49828.1 Bbu_N40 gb|ACS94765.1Bga_N34 emb|CAB64763.1 Bga_PBr (serotype 3) emb|CAA56549.1 Bbu_P0A3N6.1sp|P0A3N6.1 Bga_Nov1006 gb|ACD02016.1 Bga_PBi (serotype 4)emb|CAA56550.1 Bbu_PBo emb|CAA56468.1 Bga_Nov105 gb|ABF29551.1 Bbu_PHei(serotype 5) tr|Q06228 Bbu_PBre emb|CAA59742.1 Bga_Nov14506gb|ACD02013.1 Bbu_DK29 (serotype 6) emb|CAA45010.1 Bbu_PHeiemb|CAA56544.1 Bga_Nov14606 gb|ACD02017.1 Bga_T25 (serotype 7)emb|CAA56547.1 Bbu_PKa emb|CAA56467.1 Bga_Nov2005 gb|ABF29553.1Baf_ACA-1 gb|ACJ73559.1 Bbu_PKo emb|CAA46550.1 Bga_Nov2006 gb|ACD02018.1Baf_K78 (sequenced) Bbu_Poti_B1 emb|CAB64754.1 Bga_Nov3305 gb|ABF29554.1Baf_Khab_625 gb|AAR96311.1 Bbu_Poti_B2 emb|CAB64755.1 Bga_Nov405gb|ABF29552.1 Baf_Khab2-Sakh gb|AAP94134.1 Bbu_Poti_B3 emb|CAB64756.1Bga_Nov7006 gb|ACD02014.1 Baf_Khab470 gb|AAO91923.1 Bbu_PTroemb|CAA56471.1 Bga_Nov9906 gb|ACD02015.1 Baf_Khab505 gb|AAO91925.1Bbu_PWudI emb|CAA56469.1 Bga_PBi gb|AAT93773.1 Baf_LU192 (sequenced,partial) Bbu_PWudI/6 emb|CAA56470.1 Bga_PBr emb|CAA56549.1 Baf_Mng3602gb|ABF29573.1 Bbu_PWudII emb|CAA56546.1 Bga_Q1H1H6 gb|ABF29564.1Baf_Mng4302 gb|ABF29574.1 Bbu_Q04851.1 sp|Q04851.1 Bga_T25emb|CAA56547.1 Baf_Mng6702 gb|ABF29578.1 Bbu_Q04968.1 sp|Q04968.1Bga_TIsl emb|CAA59727.1 Baf_Mng702 gb|ABF29572.1 Bbu_Q09086.1sp|Q09086.1 Bga_TN emb|CAA56545.1 Baf_Nov1105 gb|ABF29569.1 Bbu_Q09087.1sp|Q09087.1 Bga_Tom1003 gb|ABF29564.1 Baf_Nov11506 gb|ACD02019.1Bbu_Q44738 tr|Q44738 Bga_Tom1805 gb|ABF29567.1 Baf_Nov3005 gb|ABF29570.1Bbu_Q44956 emb|CAA56937.1 Bga_Tom203 gb|ABF29562.1 Baf_P0A3N7.1sp|P0A3N7.1 Bbu_Q44962 dbj|BAA06133.1 Bga_Tom2903 gb|ABF29565.1 Baf_PHoemb|CAA59724.1 Bbu_Q45039 emb|CAR95556.1 Bga_Tom3005 gb|ABF29568.1Baf_PKo gb|ABH02138.1 Bbu_Q45040 tr|Q45040 Bga_Tom303 gb|ABF29563.1Baf_PLe emb|CAA59970.1 Bbu_S-1-10 gb|AAB96354.1 Bga_Tom3101gb|ABF29557.1 Baf_PLj7 emb|CAA59725.1 Bbu_T.R.O. emb|CAA46549.1Bga_Tom3803 gb|ABF29566.1 Baf_PLud emb|CAA59726.1 Bbu_T255emb|CAA59730.1 Bga_Tom5102 gb|ABF29560.1 Baf_Tom1103 gb|ABF29581.1Bbu_UK emb|CAB64758.1 Bga_Tom5202 gb|ABF29561.1 Baf_Tom1303gb|ABF29582.1 Bbu_VS116 emb|CAB64757.1 Bga_Tom7105 gb|ABF29556.1Baf_Tom1503 gb|ABF29583.1 Bbu_VS461 emb|CAA82329.1 Bga_VS100emb|CAB64765.1 Baf_Tom2303 gb|ABF29584.1 Bbu_WI91-23 ref|ZP_03091138.1Bga_VS307 emb|CAB64764.1 Baf_Tom2403 gb|ABF29585.1 Bbu_ZQ1emb|CAA01704.1 Bga_WABSou emb|CAA59728.1 Baf_Tom2504 gb|ABF29577.1Bbu_ZS7 gb|ACK74228.1 Bja_Cow611 emb|CAB64759.1 Baf_Tom2803gb|ABF29586.1 Bga_BgVir-1 gb|ABF29555.1 Bja_F63 emb|CAB64760.1Baf_Tom3401 gb|ABF29571.1 Bga_Far04 ref|ZP_03328706.1 Bja_HO14emb|CAB64762.1 Baf_Tom3703 gb|ABF29587.1 Bga_FujiP2 gb|AAA92301.1Bja_IKA2 emb|CAB64761.1 Baf_Tom4703 gb|ABF29588.1 Bga_IP90emb|CAJ75754.1 Blu_A8D057 gb|ABR22627.1 Baf_Tom5403 gb|ABF29575.1Bga_Ip90 emb|CAJ75754.1 Blu_A8D060 gb|ABR22625.1 Baf_Tom603gb|ABF29579.1 Bga_JEM1 gb|AAB81567.1 Blu_A8D075 gb|ABR22628.1Baf_Tom6303 gb|ABF29576.1 Bga_JEM2 gb|AAB81569.1 Blu_A8D079gb|ABR22629.1 Baf_Tom703 gb|ABF29580.1 Bga_JEM3 gb|AAB81571.1Blu_ABR22624.1 gb|ABR22624.1 Baf_XJ23 gb|AAB95225.1 Bga_JEM4dbj|BAA19222.1 Blu_ABR22626.1 gb|ABR22626.1 Bbu_118a ref|ZP_02720644.1Bga_JEM5 gb|AAB81573.1 Bsp_A14S gb|AAD16455.1 Bbu_156a gb|ACL33776.1Bga_JEM6 gb|AAB81575.1 Btu_Ya501 dbj|BAA32513.1 Bbu_19857 emb|CAA48196.1Bga_JEM7 gb|AAB81577.1 Bva_AR-2 gb|AAF00571.1 Bbu_2005348A prf|2005348ABga_JEM8 gb|AAB81579.1 Bva_M19 gb|AAF00573.1 Bbu_2005348B prf|2005348BBga_Khab3155 gb|AAR96310.1 Bva_M49 gb|AAF00574.1 Bbu_297 emb|CAA59729.1Bga_Khab550 gb|AAR96306.1 Bva_M52 gb|AAF00575.1 Bbu_29805ref|ZP_03092996.1 Bga_Khab616 gb|AAR96307.1 Bva_M53 gb|AAF00576.1Bbu_64b ref|ZP_03097520.1 Bga_Khab648 gb|AAR96308.1 Bva_M7 gb|AAF00572.1Bbu_72a ref|ZP_02724465.1 Bga_Khab722 gb|AAR96309.1 Bva_Q9RM88emb|CAB56150.1 Bbu_80a ref|ZP_03088001.1 Bga_Khab23 gb|AAP94125.1Bva_QLZSP1 gb|ACA13516.1 Bbu_94a ref|ZP_02725946.1 Bga_Khab24gb|AAP94126.1 Bva_QSDS4 gb|ACA13517.1 Bbu_AAB23809.1 gb|AAB23809.1Bga_Khab31 gb|AAP94127.1 Bva_QSYSP3 gb|ACA13518.1 Bbu_AAB23810.1gb|AAB23810.1 Bga_Khab31a gb|AAP94128.1 Bva_QSYSP4 gb|ACA13519.1 Bbu_B29gb|AAA18508.1 Bga_Khab-466 gb|AAP94129.1 Bva_QTMP2 gb|ACA13520.1 Bbu_B31gb|AAC66260.1 Bga_Khab489 gb|AAP94130.1 Bva_QX-S13 gb|ACA13521.1Bbu_Bol26 ref|ZP_02531917.1 Bga_Khab5-Sakh gb|AAO91932.1 Bva_UKgb|AAF00570.1 Bbu_C-1-11 gb|AAB96351.1 Bga_Khab506 gb|AAP94132.1Bva_VS116 gb|AAF00569.1 Bbu_CA-11.2a_1 ref|ZP_03094587.1 Bga_Khab516gb|AAP94133.1 Bsp_10MT dbj|BAA32516.1 Bbu_CA-11.2a_2 ref|ZP_03094587.1Bga_Khab721 gb|AAP94131.1 Bsp_5MT dbj|BAA32515.1 Bbu_CA-11.2a_CA-112aref|ZP_03094587.1 Bga_Khab2119 gb|AAO91928.1 Bsp_Am501 dbj|BAA32514.1Bbu_CAA00316.1 emb|CAA00316.1 Bga_Khab2559 gb|AAO91929.1 Bsp_LV5gb|AAB96353.1 Bbu_CAA42842.1 emb|CAA42842.1 Bga_Khab2560 gb|AAO91930.1Bsp_PAnz emb|CAJ43585.1 Bbu_CAA44258.1 emb|CAA44258.1 Bga_Khab2594gb|AAO91931.1 Bsp_PHaP_PHap emb|CAJ43582.1 Bbu_CAR95597.1 emb|CAR95597.1Bga_Khab430 gb|AAO91919.1 Bsp_PJes emb|CAJ43586.1 Bbu_DK1 gb|AAA22955.1Bga_Khab448 gb|AAO91920.1 Bsp_PMai emb|CAJ43584.1 Bbu_DK29emb|CAA45010.1 Bga_Khab457 gb|AAO91921.1 Bsp_PMew emb|CAJ43583.1Bbu_DK6_Danish_isolate emb|CAA58601.1 Bga_Khab468 gb|AAO91922.1Bsp_PSigII emb|CAJ43581.1 Bbu_G2 gb|AAA88846.1 Bga_Khab492 gb|AAO91924.1Bsp_SV1 ref|ZP_03095680.1 Bbu_G25 emb|CAA82328.1 Bga_Khab511gb|AAO91926.1 Bbi_25015 gb|AAB21761.1 Bbu_H.E. emb|CAA46551.1Bga_Khab560 gb|AAO91927.1 Bbi_DN127 emb|CAB64766.1 Bbu_HB19gb|AAC18776.1 Bga_LV4 gb|AAB96352.1 Bbi_Q09087.1 gb|AAB21761.1Abbreviations: Baf = Borrelia afzelii, Bbu = Borrelia burgdorferi s.s.,Bga = Borrelia garinii, Bsp = Borrelia spielmanii, Bbi = Borreliabissettii, Bva = Borrelia valaisiana, Btu = Borrelia turicatae, Bdu =Borrelia duttonii, Blu = Borrelia lusitaniae, Bja = Borrelia japonica,gb = GenBank, emb = EMBL, tr = UniProt/tremble, sp = UniProt/Swissprot,prf = Protein Research Foundation, dbj = DNA Databank of Japan (DDBJ),pdb = Protein Data Bank, db = database

In accordance with the present invention, the disulfide bond may beformed between cysteines that have been introduced at any position ofthe OspA fragment allowing or supporting appropriate folding of thefragment. The positions may be selected, as detailed above, based on theknown structure of the OspA. In a preferred embodiment, the polypeptideof the current invention contains at least one disulfide bond betweenany of positions 182+/−3 and any of positions 269+/−3 (disulfide bondtype 1); any of positions 182+/−3 and any of positions 272+/−3(disulfide bond type 2); any of positions 244+/−3 and any of positions259+/−3 (disulfide bond type 3); any of positions 141+/−3 and any ofpositions 241+/−3 (disulfide bond type 4); any of positions 165+/−3 andany of positions 265+/−3 (disulfide bond type 5); any of positions185+/−3 and any of positions 272+/−3 (disulfide bond type 6); any ofpositions 199+/−3 and any of positions 223+/−3 (disulfide bond type 7);any of positions 243+/−3 and any of positions 262+/−3 (disulfide bondtype 8); any of positions 184+/−3 and any of positions 204+/−3(disulfide bond type 9); any of positions 201+/−3 and any of positions214+/−3 (disulfide bond type 10); any of positions 246+/−3 and any ofpositions 259+/−3 (disulfide bond type 11); and/or any of positions167+/−3 and any of positions 178+/−3 (disulfide bond type 12) of a B.afzelii, particularly B. afzelii K78 serotype 2 OspA, or the homologousamino acids of an OspA from a Borrelia sp. other than B. afzelii, suchas B. burgdorferi s.s., particularly strain B31, serotype 1; B. garinii,particularly strain PBr, serotype 3; B. bavariensis, particularly strainPBi, serotype 4; B. garinii, particularly strain PHei, serotype 5; B.garinii, particularly strain DK29, serotype 6 or B. garinii,particularly strain T25, serotype 7.

More particularly, the polypeptide of the current invention contains theat least one disulfide bond between any of positions 182 and 269(disulfide bond type 1); positions 182 and 272 (disulfide bond type 2);positions 244 and 259 (disulfide bond type 3); positions 141 and 241(disulfide bond type 4); positions 165 and 265 (disulfide bond type 5);positions 185 and 272 (disulfide bond type 6); positions 199 and 223(disulfide bond type 7); positions 243 and 262 (disulfide bond type 8);positions 184 and 204 (disulfide bond type 9); positions 201 and 214(disulfide bond type 10); positions 246 and 259 (disulfide bond type11); and/or positions 167 and 178 (disulfide bond type 12) of a B.afzelii, particularly B. afzelii K78 serotype 2 OspA, or the homologousamino acids of an OspA from a Borrelia other than B. afzelii, such as B.burgdorferi s.s., particularly strain B31, serotype 1; B. garinii,particularly strain PBr, serotype 3; B. bavariensis, particularly strainPBi, serotype 4; B. garinii, particularly strain PHei, serotype 5; B.garinii, particularly strain DK29, serotype 6 or B. garinii,particularly strain T25, serotype 7.

TABLE A-4 Disulfide bond types with nomenclature and the position of thecysteine substitutions in the serotype 2 OspA protein. Position ofcysteines in B. Disulfide bond type Nomenclature afzelii K78 serotype 2OspA wild-type sequence D0 No cysteine substitutions 1 D1 182 and 269 2D2 182 and 272 3 D3 244 and 259 4 D4 141 and 241 5 D5 165 and 265 6 D6185 and 272 7 D7 199 and 223 8 D8 243 and 262 9 D9 184 and 204 10 D10201 and 214 11 D11 246 and 259 12 D12 167 and 178

Even more preferred are disulfide bond types 1 to 5, especiallydisulfide bond types 1 to 4.

It is noted that:

-   -   Position 182+/−3 is an abbreviation for position 179, 180, 181,        182, 183, 184 or 185, preferably 182.    -   Position 269+/−3 is an abbreviation for position 266, 267, 268,        269, 270, 271 or 272, preferably 269.    -   Position 272+/−3 is an abbreviation for position 269, 270, 271,        272, 273, 274 or 275, preferably 272.    -   Position 244+/−3 is an abbreviation for position 241, 242, 243,        244, 245, 246 or 247, preferably 244.    -   Position 259+/−3 is an abbreviation for position 256, 257, 258,        259, 260, 261 or 262, preferably 259.    -   Position 141+/−3 is an abbreviation for position 138, 139, 140,        141, 142, 143 or 144, preferably 141.    -   Position 241+/−3 is an abbreviation for position 238, 239, 240,        241, 242, 243 or 244, preferably 241.    -   Position 165+/−3 is an abbreviation for position 162, 163, 164,        165, 166, 167 or 168, preferably 165.    -   Position 265+/−3 is an abbreviation for position 262, 263, 264,        265, 266, 267 or 268, preferably 265.    -   Position 185+/−3 is an abbreviation for position 182, 183, 184,        185, 186, 187 or 188, preferably 185.    -   Position 199+/−3 is an abbreviation for position 196, 197, 198,        199, 200, 201 or 202, preferably 199.    -   Position 223+/−3 is an abbreviation for position 220, 221, 222,        223, 224, 225 or 226, preferably 223.    -   Position 243+/−3 is an abbreviation for position 240, 241, 242,        243, 244, 245 or 246, preferably 143.    -   Position 262+/−3 is an abbreviation for position 259, 260, 261,        262, 263, 264 or 265, preferably 262.    -   Position 184+/−3 is an abbreviation for position 181, 182, 183,        184, 185, 186 or 187, preferably 184.    -   Position 204+/−3 is an abbreviation for position 201, 202, 203,        204, 205, 206 or 207, preferably 204.    -   Position 201+/−3 is an abbreviation for position 198, 199, 200,        201, 202, 203 or 204, preferably 201.    -   Position 214+/−3 is an abbreviation for position 211, 212, 213,        214, 215, 216 or 217, preferably 214.    -   Position 246+/·3 is an abbreviation for position 243, 244, 245,        246, 247, 248 or 249, preferably 246.    -   Position 167+/−3 is an abbreviation for position 164, 165, 166,        167, 168, 169 or 170, preferably 167.    -   Position 178+/−3 is an abbreviation for position 175, 176, 177,        178, 179, 180 or 181, preferably 178.

In a preferred embodiment, the mutant fragment is derived from the aminoacids from position 126, 130 or 13 1 to position 273 of the wild-typesequence of the OspA of B. afzelii strain K78, serotype 2 (SEQ ID NO:18) and differs only by the introduction of at least one disulfide bond,particularly wherein the at least one disulfide bond is betweenpositions 182 and 269 (disulfide bond type 1); positions 182 and 272(disulfide bond type 2); positions 244 and 259 (disulfide bond type 3);positions 141 and 241 (disulfide bond type 4); positions 165 and 265(disulfide bond type 5); positions 185 and 272 (disulfide bond type 6);positions 199 and 223 (disulfide bond type 7); positions 243 and 262(disulfide bond type 8); positions 184 and 204 (disulfide bond type 9);positions 201 and 214 (disulfide bond type 10); positions 246 and 259(disulfide bond type 11); and/or positions 167 and 178 (disulfide bondtype 12), or the homologous fragments and positions of an OspA from aBorrelia sp. other than B. afzelii, such as B. burgdorferi s.s.,particularly strain B31, serotype 1; B. garinii, particularly strainPBr, serotype 3; B. bavariensis, particularly strain PBi, serotype 4; B.garinii, particularly strain PHci, serotype 5; B. garinii, particularlystrain DK29, serotype 6 or B. garinii, particularly strain T25, serotype7.

In a still more preferred embodiment, the mutant fragment has an aminoacid sequence selected from the group consisting of SEQ ID NO: 167, SEQID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQID NO: 177, SEQ ID NO: 178 and an amino acid sequence that has 80%, morepreferably 85%, more preferably 90%, even more preferably 95% sequenceidentity to at least one of sequences with SEQ ID NOs: 2 to 13, whereinthe cysteines are not replaced. Further details on mutations andsequence identity are given above.

As detailed above, the polypeptide of the present invention may comprisesignal sequences. It has been shown that lipidation confers adjuvantproperties on OspA. Accordingly, lipidated forms of the polypeptide ofthe invention or polypeptides comprising a lipidation signal arepreferred. In a preferred embodiment, the polypeptide of the currentinvention comprises a lipidation signal, preferably a lipidation signalof a Borrelia outer surface protein, OspA or OspB (SEQ ID NOs: 14 and15, respectively) or more preferably an E. coli lpp lipidation signalsequence (SEQ ID NO: 16). The OspA fragment of the invention comprisinga lipidation signal is lipidated during processing and the lipidationsignal peptide is cleaved off. Therefore the signal peptide is no longerpresent in the mature lipidated protein.

Lipidated proteins according to the current invention are labeled with“Lip” at the N-terminus to indicate the addition of 3 fatty acid groupsand a glycerol to the polypeptide (sec FIG. 4 ). Suitable lipidationsignals as described above include MKKYLLGIGLILALIA (SEQ ID NO: 14),MRLLIGFALALALIG (SEQ ID NO: 15) and MKATKLVLGAVILGSTLLAG (SEQ ID NO:16). Because lipid moieties and a glycerol are attached to theN-terminal cysteine residue which is present in the full-lengthwild-type OspA protein, OspA C-terminal fragments for lipidation mayadditionally comprise a peptide comprising a cysteine residue followedby additional amino acids, herein referred to as “Lipidation Peptide” or“LP” (see FIGS. 1 and 2 ). For example, sequences such as CSS or CKQN(SEQ ID NO: 211) immediately C-terminal to the lipidation signalsequence provide an N-terminal cysteine residue for lipidation uponcleavage of the lipidation signal peptide. The lipidatedcysteine-containing peptides are present in the final lipidatedpolypeptide of the invention.

It has been found that the OspA protein of B. burgdorferi s.s. comprisesa sequence with the capacity to bind to a T-cell receptor that also hasthe capacity to bind to human leukocyte function-associated antigen(hLFA-1) (herein referred to also as “hLFA-1-like sequence”). Thesimilarity of this OspA region to hLFA-1 may result in an immuneresponse with cross-reactivity upon administration of B. burgdorferis.s. OspA to a human subject and may induce autoimmune diseases,particularly autoimmune arthritis, in susceptible individuals.Accordingly, in a preferred embodiment, the polypeptide of the currentinvention does not comprise a sequence with binding capacity to theT-cell receptor that has a binding capacity to the human leukocytefunction-associated antigen (hLFA-1), and particularly does not comprisethe amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17). To this end, thehLFA-1-like sequence, particularly the amino acid sequence GYVLEGTLTAE(SEQ ID NO: 17), may be replaced with a homologous sequence from an OspAprotein of another Borrelia sp., particularly with NFTLEGKVAND (SEQ IDNO: 18).

In a preferred embodiment, the polypeptide of the current inventioncomprising at least one disulfide bond essentially establishes the sameprotective capacity with said polypeptide against a Borrelia infectionrelative to at least one of the wild-type full-length OspA proteinsderived from at least one Borrelia strain, particularly B. afzelii K78,OspA serotype 2 (SEQ ID NO: 19); B. burgdorferi s.s., particularlystrain B31, serotype 1 (SEQ ID NO: 20); B. garinii, particularly strainPBr, serotype 3 (SEQ ID NO: 21); B. bavariensis, particularly strainPBi, serotype 4 (SEQ ID NO: 22); B. garinii, particularly strain PHei,serotype 5 (SEQ ID NO: 23); B. garinii, particularly strain DK29,serotype 6 (SEQ ID NO: 24) or B. garinii, particularly strain T25,serotype 7 (SEQ ID NO: 25).

In order to provide cross-protection against different Borrelia speciesor OspA serotypes, the development of a multivalent vaccine isdesirable. Accordingly, in another preferred embodiment, the polypeptideof the first aspect comprises at least two mutant fragments from twodifferent Borrelia serotypes as defined above. In a preferredembodiment, the polypeptide of the first aspect comprises at least twomutant OspA fragments which are selected from the group consisting of

-   -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 2;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 3;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 3;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 10;    -   figment with disulfide bond type 2 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 10 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 10 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 11 and fragment with disulfide        bond type 12;    -   and        particularly wherein    -   the fragment with disulfide bond type 1 has the amino acid        sequence of SEQ ID NO: 2 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ 1D NO: 2, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 2 has the amino acid        sequence of SEQ ID NO: 3 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 3, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 3 has the amino acid        sequence of SEQ ID NO: 4 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 4, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 4 has the amino acid        sequence of SEQ ID NO: 5 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 5, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 5 has the amino acid        sequence of SEQ ID NO: 6 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 6, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 6 has the amino acid        sequence of SEQ ID NO: 7 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 7, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 7 has the amino acid        sequence of SEQ ID NO: 8 or an amino acid sequence that has at        least 80% more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 8, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 8 has the amino acid        sequence of SEQ ID NO: 9 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 9, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 9 has the amino acid        sequence of SEQ ID NO: 10 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 10, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 10 has the amino acid        sequence of SEQ ID NO: 11 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 11, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 11 has the amino acid        sequence of SEQ ID NO: 12 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 12, wherein the        cysteines are not replaced; and/or    -   the fragment with disulfide bond type 12 has the amino acid        sequence of SEQ ID NO: 13 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 13, wherein the        cysteines are not replaced.

Please note that further details on mutations and sequence identity aregiven above.

TABLE A-5 Nomenclature and SEQ ID NOs. of mutant OspA fragmentheterodimers, non-lipidated and lipidated, described in the currentinvention. SEQ ID NO: Mutant OspA fragment heterodimer* S1D4-S2D4 43S1D1-S2D1 47 S3D4-S4D4 51 S3D1-S4D1 55 S5D4-S6D4 59 S5D1-S6D1 63S2D4-S1D4 67 S2D1-S1D1 71 S4D4-S3D4 75 S4D1-S3D1 79 S6D4-S5D4 83S6D1-S5D1 87 S1D4-S2D1 91 S1D1-S2D4 95 S3D4-S4D1 99 S3D1-S4D4 103S5D4-S6D1 107 S5D1-S6D4 111 S2D4-S1D1 115 S2D1-S1D4 119 S4D4-S3D1 123S4D1-S3D4 127 S6D4-S5D1 131 S6D1-S5D4 135 Lipidated mutant OspA fragmentheterodimer* Lip-S1D4-S2D4 185 Lip-S1D1-S2D1 186 Lip-S3D4-S4D4 187Lip-S3D1-S4D1 188 Lip-S5D4-S6D4 189 Lip-S5D1-S6D1 190 Lip-S2D4-S1D4 191Lip-S2D1-S1D1 192 Lip-S4D4-S3D4 193 Lip-S4D1-S3D1 194 Lip-S6D4-S5D4 195Lip-S6D1-S5D1 196 Lip-S1D4-S2D1 197 Lip-S1D1-S2D4 198 Lip-S3D4-S4D1 199Lip-S3D1-S4D4 200 Lip-S5D4-S6D1 201 Lip-S5D1-S6D4 202 Lip-S2D4-S1D1 203Lip-S2D1-S1D4 204 Lip-S4D4-S3D1 205 Lip-S4D1-S3D4 206 Lip-S6D4-S5D1 207Lip-S6D1-S5D4 208 *S = Serotype (1-6) (see Table A-2); D = DisulfideBond Type (see Table A-4); Lip = lipidation: the N-terminal addition ofglycerol and fatty acid residues.

In another preferred embodiment, the polypeptide according to the firstaspect comprises at least two or three mutant fragments which areconnected via one or more linkers. A linker is a rather short amino acidsequence employed to connect two fragments. It should be designed inorder to avoid any negative impact on the fragments, their interactionin subjects to be treated or vaccinated or upon their protectivecapacity. Preferred are short linkers of at most 21 amino acids,particularly at most 15 amino acids, especially at most 12 or 8 aminoacids. More preferably, the one or more linkers is/are composed of smallamino acids in order to reduce or minimize interactions with thefragments, such as glycine, serine and alanine. Examples or preferredlinkers include linkers comprising or consisting of polyG, such as (G)₈(SEQ ID NO: 36) (G)₁₂ (SEQ ID NO: 37), GAGA (SEQ ID NO: 38), (GAGA)₂(SEQ ID NO: 39), (GAGA)₃ (SEQ ID NO: 40), (GGGS)₂ (SEQ ID NO: 41), or(GGGS)₃ (SEQ ID NO: 42). A more preferred linker is the “LN1” peptidelinker, a fusion of two separate loop regions of the N-terminal half ofOspA from B. burgdorferi s.s., strain B31 (aa 65-74 and aa 42-53, withan amino acid exchange at position 53 of D53S) which has the followingsequence:

(SEQ ID NO: 184) GTSDKNNGSGSKEKNKDGKYS.

In another preferred embodiment, the polypeptide according to the firstaspect comprises a polypeptide with a total size of at most 500 aminoacids, comprising two or three different mutant fragments as defined inpreferred embodiments of the first aspect; or a polypeptide whichconsists of essentially two or three different mutant fragments, one ortwo linkers and, optionally, an N-terminal cysteine; and/or apolypeptide which consists of essentially two or three different mutantfragments, an N-terminal extension of the fragment consisting of at most24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acids,preferably at most 10, 9, 8, 7 or 6 amino acids, still more preferablyat most 5, 4, 3, 2 or 1 amino acid(s), wherein the N-terminal extensionis located directly N-terminally from the fragment in the respectiveBorrelia OspA and, optionally, an N-terminal cysteine. The N-terminalcysteine may optionally be followed by a short peptide linker from 1-10amino acids long, and preferably takes the form of an N-terminal CSSpeptide or CKQN peptide (SEQ ID NO: 211).

In a second aspect, the present invention relates to a nucleic acidencoding for the polypeptide according to the first aspect.

The invention further provides a nucleic acid encoding a polypeptide ofthe invention. For the purposes of the invention the term “nucleicacid(s)” generally refers to any polyribonucleotide orpolydeoxynucleotide, which may be unmodified RNA or DNA or modified RNAor DNA including single and double-stranded regions/forms.

The term “nucleic acid encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding a peptideor polypeptide of the invention. The term also encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the peptide or polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal similarity to the nucleotide sequence ofany native (i.e., naturally occurring) gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention, for examplepolynucleotides that are optimized for human and/or primate and/or E.coli codon selection.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. of al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al.,Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge et al., Science 269:202-204 (1995)) and automated synthesis maybe achieved, for example, using the ASI 431 A Peptide Synthesizer(Perkin Elmer, Palo Alto, CA).

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences, inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In a further aspect of the invention the present invention relatesvector comprising a nucleic acid of the invention linked to an induciblepromoter such that when the promoter is induced a polypeptide encoded bythe nucleic acid is expressed. In a preferred embodiment, the vector ispET28b(+).

A further aspect of the invention comprises said vector wherein theinducible promoter is activated by addition of a sufficient quantity ofIPTG (Isopropyl β-D-1-thiogalactopyranoside) preferably to the growthmedium. Optionally this is at a concentration of between 0.1 and 10 mM,0.1 and 5 mM, 0.1 and 2.5 mM, 0.2 and 10 mM, 0.2 and 5 mM, 0.2 and 2.5mM, 0.4 and 10 mM, 1 and 10 mM, 1 and 5 mM, 2.5 and 10 mM, 2.5 and 5 mM,5 and 10 mM. Alternatively the promoter may be induced by a change intemperature or pH.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to at least single- and double-strandedDNA, hybrid molecules comprising DNA and RNA that may be single-strandedor, more typically, double-stranded, or a mixture of single- anddouble-stranded regions. As used herein, the term nucleic acid moleculeincludes DNA or RNA molecules as described above that contain one ormore modified bases. Thus, DNA or RNA molecules with backbones modifiedfor stability or for other reasons are “nucleic acid molecule” as thatterm is intended herein. Moreover, DNA or RNA species comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are also nucleic acid molecules as definedherein. It will be appreciated that a great variety of modificationshave been made to DNA and RNA molecules that serve many useful purposesknown to those of skill in the art. The term nucleic acid molecule asused herein embraces such chemically, enzymatically or metabolicallymodified forms of nucleic acid molecules, as well as the chemical formsof DNA and RNA characteristic of viruses and cells, including simple andcomplex cells, inter alia. The term nucleic acid molecule alsoencompasses short nucleic acid molecules often referred to asoligonucleotide(s). The terms “polynucleotide” and “nucleic acid” or“nucleic acid molecule” are used interchangeably herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated fromBorrelia and modified by methods known to one skilled in the art. Thesame applies to the polypeptides according to the present invention.

Furthermore, the nucleic acid of the present invention can befunctionally linked, using standard techniques such as cloning, to anydesired sequence(s), whether a Borrelia regulatory sequence or aheterologous regulatory sequence, heterologous leader sequence,heterologous marker sequence or a heterologous coding sequence to createa fusion gene.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthesis techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand. Thenucleic acid of the present invention may be comprised in a vector or ina cell. The vector may comprise the above-mentioned nucleic acid in sucha manner that the vector is replicable and can express the proteinencoded by the nucleotide sequence in a host cell.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or nucleic acid of the invention. Introduction of anucleic acid into the host cell can be effected by methods described inmany standard laboratory manuals, such as Davis, et al., BASIC METHODSIN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, conjugation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include gram negativebacterial cells, such as cells of E. coli, Acinetobacter,Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria,Enterobacter, Erwinia, Franciscella, Helicobacter, Hemophilus,Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus,Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio,Yersinia. In one embodiment the host cell is an Escherichia coli cell.In a preferred embodiment, the host cell is an E. coli BL21 (DE3) cellor an E. coli BL21 Star™ (DE3) cell.

Alternatively gram positive bacterial cells may also be used. A greatvariety of expression systems can be used to produce the polypeptides ofthe invention. In one embodiment the vector is derived from bacterialplasmids. Generally any system or vector suitable to maintain, propagateor express polynucleotides and/or to express a polypeptide in a host maybe used for expression in this regard. The appropriate DNA sequence maybe inserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

In one embodiment of the current invention, the cells are gown underselective pressure, such as in the presence of antibiotics, preferablykanamycin. In another embodiment, cells are grown in the absence ofantibiotics.

A great variety of expression vectors can be used to express thepolypeptides according to the present invention. Generally, any vectorsuitable to maintain, propagate or express nucleic acids to express apolypeptide in a host may be used for expression in this regard. Inaccordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single- or double-stranded phage vector ora single- or double-stranded RNA or DNA viral vector. Starting plasmidsdisclosed herein are either commercially available, publicly available,or can be constructed from available plasmids by routine application ofwell-known, published procedures. Preferred among vectors, in certainrespects, are those for expression of nucleic acid molecules and thepolypeptides according to the present invention. Nucleic acid constructsin host cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thepolypeptides according to the present invention can be syntheticallyproduced by conventional peptide synthesizers.

In addition, the present invention relates to a host cell comprisingthis vector. Representative examples of appropriate host cells includebacteria, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis; fungi, such as yeast and Aspergillus; insect cellssuch as Drosophila S2 and Spodoptera Sf9 cells; mammalian cells such asCHO, COS, HeLa, C127, 3T3, BHK, 293 or Bowes melanoma cells; and plantcells. Cell-free translation systems can also be employed to producesuch proteins using RNA derived from the DNA construct of the presentinvention.

In order to express the desired amino acid sequence practically byintroducing the vector according to the present invention into a hostcell, the vector may contain, in addition to the nucleic acid sequenceaccording to the present invention, other sequences for controlling theexpression (e.g., promoter sequences, terminator sequences and enhancersequences) and gene markers for selecting microorganisms, insect cells,animal culture cells, or the like (e.g., neomycin resistance genes andkanamycin resistance genes). Furthermore, the vector may contain thenucleic acid sequence according to the present invention in a repeatedform (e.g., in tandem). The vector may be constructed based onprocedures and manners which are conventionally used in the field ofgenetic engineering.

The host cells may be cultured in an appropriate medium, and the proteinaccording to the present invention may be obtained from the cultureproduct. The protein according to the present invention may be recoveredfrom the culture medium and purified in the conventional manner.

The problem underlying the present invention is furthermore solved by amethod for producing a polypeptide as defined above, characterized bythe following steps:

-   -   a) introducing a vector encoding the polypeptide into a host        cell,    -   b) growing the host cell under conditions allowing for        expression of said polypeptide,    -   c) homogenizing said host cell, and    -   d) subjecting the host cell homogenate to purification steps.

The invention further relates to a method for producing a polypeptide asdefined above, characterized by the following steps:

-   -   a) introducing a nucleic acid encoding a polypeptide into a        vector,    -   b) introducing said vector into a host cell,    -   c) growing said host cell under conditions allowing for        expression of polypeptide,    -   d) homogenizing said host cell.    -   e) enriching polypeptide in the lipid phase by phase separation,        and    -   f) further purifying over a gel filtration column.

The invention further relates to a method for producing a polypeptide asdefined above, characterized by the following steps:

-   -   a) introducing a nucleic acid encoding a polypeptide into a        vector,    -   b) introducing said vector into a host cell,    -   c) growing said host cell under conditions allowing for        expression of polypeptide,    -   d) homogenizing said host cell.    -   e) enriching polypeptide in the lipid phase by phase separation,    -   g) purifying over a gel filtration column, and    -   h) optionally, further processing over a buffer exchange column.

The problem underlying the present invention is solved in a furtheraspect by an antibody, or at least an effective part thereof, whichspecifically binds to at least a selective part of a polypeptide, asdefined above.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment said effective part comprises an Fabfragment, an F(ab) fragment, an F(ab)N fragment, an F(ab)₂ fragment oran F_(v) fragment.

In still another embodiment of the invention the antibody is a chimericantibody.

In yet another embodiment the antibody is a humanized antibody.

In a preferred aspect, antibodies of the invention bind specifically tomutant OspA fragment polypeptides of the invention, but not tocorresponding wild-type OspA fragment polypeptides. In a inure preferredaspect, the antibody binds specifically to the disulfide bond of themutant OspA fragment of the invention.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as a Nanobody or a polypeptideof the invention) molecule can bind. The specificity of anantigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (K_(D)), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)).

As will be clear to the skilled person (for example on the basis of thefurther disclosure herein), affinity can be determined in a manner knownper se, depending on the specific antigen of interest. Avidity is themeasure of the strength of binding between an antigen-binding molecule(such as an antibody or an effective part thereof of the invention) andthe pertinent antigen. Avidity is related to both the affinity betweenan antigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule. Typically, antigen-bindingproteins (such as an antibody or an effective part thereof of theinvention) will bind to their antigen with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter(i.e. with an association constant (K_(A)) of 10⁵ to 10¹² liter/moles ormore, and preferably 10⁷ to 10¹² liter/moles or more and more preferably10⁸ to 10¹² liter/moles). Any K_(D) value greater than 10⁴ mol/liter (orany K_(A) value lower than 10⁴ M⁻¹) liters/mol is generally consideredto indicate non-specific binding. Preferably, a monovalentimmunoglobulin sequence of the invention will bind to the desiredantigen with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM. Specificbinding of an antigen-binding protein to an antigen or antigenicdeterminant can be determined in any suitable manner known per se,including, for example, Scatchard analysis and/or competitive bindingassays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) andsandwich competition assays, and the different variants thereof knownper se in the art, as well as the other techniques mentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more than 10⁴ moles/liter or 10³ moles/liter(e.g., of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(K_(A)), by means of the relationship [K_(D)=1/K_(A)].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the K_(D), ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, K_(A), which equals1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two molecules(such as an amino acid sequence, Nanobody or polypeptide of theinvention and its intended target) will mainly be expressed in terms ofthe K_(D) value of their interaction; it being clear to the skilledperson that in view of the relation K_(A)=1/K_(D), specifying thestrength of molecular interaction by its K_(D) value can also be used tocalculate the corresponding K_(A) value. The K_(D) value characterizesthe strength of a molecular interaction also in a thermodynamic sense asit is related to the free energy (DG) of binding by the well knownrelation DG=RT·ln(K_(D)) (equivalently DG=−RT·ln(K_(A))), where R equalsthe gas constant, T equals the absolute temperature and In denotes thenatural logarithm.

The K_(D) for biological interactions which are considered meaningful(e.g. specific) are typically in the range of 10⁻¹⁰ M (0.1 nM) to 10⁻⁵ M(10000 nM). The stronger an interaction, the lower its K_(D).

In a preferred embodiment, the K_(D) of the antibody of the invention isbetween 10⁻¹² M and 10⁻⁵ M, preferably less than 10⁻⁶, preferably lessthan 10⁻⁷, preferably less than 10⁻⁸ M, preferably less than 10⁻⁹ M,more preferably less than 10⁻¹⁰ M, even more preferably less than 10⁻¹¹M, most preferably less than 10⁻¹² M.

The K_(D) can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as k_(off), to the rate of itsassociation, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe S1 unit notation for second). The on-rate k_(on) has units M⁻¹ s⁻¹.The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,approaching the diffusion-limited association rate constant forbimolecular interactions. The off-rate is related to the half-life of agiven molecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate may vary between 10⁻⁶ s⁻¹ (near irreversible complex with at_(1/2) of multiple days) to 1 s⁻¹ (t_(1/2)=0.69 s).

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern. Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(off),k_(on) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artefacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition sites for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of K_(D) may be quite labor-intensive;therefore, apparent K_(D) values are often determined in order to assessthe binding strength of two molecules. It should be noted that as longall measurements are made in a consistent way (e.g. keeping the assayconditions unchanged), apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or flow cytometry or other format(the fluorophore for fluorescence detection, the chromophore for lightabsorption detection, the biotin for streptavidin-mediated ELISAdetection). Typically, the reference molecule C is kept at a fixedconcentration and the concentration of A is varied for a givenconcentration or amount of B. As a result an Inhibitory Concentration(IC)₅₀ value is obtained corresponding to the concentration of A atwhich the signal measured for C in absence of A is halved. ProvidedK_(D ref), the K_(D) of the reference molecule, is known, as well as thetotal concentration c_(ref) of the reference molecule, the apparentK_(D) for the interaction A-B can be obtained from following formula:K_(D)=IC₅₀/(1+c_(ref)/K_(Dref)). Note that if c_(ref)<<K_(Dref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the strength or stability of a molecular interaction can beassessed by the IC₅₀ and this measurement is judged as equivalent toK_(D) or to apparent K_(D) throughout this text.

Another aspect of the invention relates to a hybridoma cell line, whichproduces an antibody as defined above.

The problem underlying the present invention is furthermore solved by amethod for producing an antibody as defined above, characterized by thefollowing steps:

-   -   a) initiating an immune response in a non-human animal by        administering a polypeptide as defined above to said animal,    -   b) removing an antibody containing body fluid from said animal,        and    -   c) producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

The invention further relates to a method for producing an antibody asdefined above, characterized by the following steps:

-   -   a) initiating an immune response in a non-human animal by        administering a polypeptide as defined above to said animal,    -   b) removing the spleen or spleen cells from said animal,    -   c) producing hybridoma cells of said spleen or spleen cells,    -   d) selecting and cloning hybridoma cells specific for said        polypeptide,    -   e) producing the antibody by cultivation of said cloned        hybridoma cells, and    -   f) optionally conducting further purification steps.

Another aspect of the present invention is related to a pharmaceuticalcomposition comprising an antibody as specified above.

Still another aspect relates to an antibody as defined above or apharmaceutical composition comprising an antibody as defined above forthe treatment or prevention of an infection with Borrelia species, morepreferably pathogenic Borrelia species as disclosed herein morepreferably comprising B. burgdorferi s.s., B. afzelii, B. bavariensisand B. garinii.

The problem underlying the present invention is solved in another aspectby the use of an antibody as defined above for the preparation of apharmaceutical composition for treating or preventing infections withBorrelia species, more preferably pathogenic Borrelia species asdisclosed herein more preferably comprising B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii.

In a third aspect the present invention relates to a pharmaceuticalcomposition comprising the polypeptide according to the first aspectand/or the nucleic acid according to the second aspect. Thepharmaceutical composition may optionally contain any pharmaceuticallyacceptable carrier or excipient, such as buffer substances, stabilisersor further active ingredients, especially ingredients known inconnection with pharmaceutical compositions and/or vaccine production.Preferably, the pharmaceutical composition is used as a medicament,particularly as a vaccine or for preventing or treating an infectioncaused by Borrelia species, more preferably pathogenic Borrelia speciesas disclosed herein more preferably comprising B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii, and/or other pathogens againstwhich the antigens have been included in the vaccine.

In one embodiment the pharmaceutical composition further comprises anadjuvant. The choice of a suitable adjuvant to be mixed with bacterialtoxins or conjugates made using the processes of the invention is withinthe knowledge of the person skilled in the art. Suitable adjuvantsinclude an aluminium salt such as aluminium hydroxide or aluminumphosphate, but may also be other metal salts such as those of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedsaccharides, or polyphosphazenes. In a preferred embodiment, thepharmaceutical composition is adjuvanted with aluminium hydroxide.

In a further embodiment, the pharmaceutical composition furthercomprises an immunostimulatory substance, preferably selected from thegroup consisting of polycationic polymers, especially polycationicpeptides, immunostimulatory oligodeoxynucleotides (ODNs), especiallyoligo(dIdC)₁₃ (SEQ ID NO: 32), peptides containing at least twoLysLeuLys motifs, especially peptide KLKLLLLLKLK (SEQ ID NO: 33),neuroactive compounds, especially human growth hormone, aluminiumhydroxide, aluminium phosphate, Freund's complete or incompleteadjuvants, or combinations thereof. Preferably, the immunostimulatorysubstance is a combination of either a polycationic polymer andimmunostimulatory deoxynucleotides or of a peptide containing at leasttwo LysLeuLys motifs and immunostimulatory deoxynucleotides, preferablya combination of KLKLLLLLKLK (SEQ ID NO: 33) and oligo(dIdC)₁₃ (SEQ IDNO: 32). More preferably, said polycationic peptide is polyarginine.

In a further embodiment, the pharmaceutical composition comprises sodiumphosphate, sodium chloride, L-methionine, sucrose and Tween-20 at a pHof 6.7+/−0.2. Preferably, the pharmaceutical composition also comprisesaluminium hydroxide, preferably at a concentration of 0.15%.

In one embodiment, the formulation comprises between 5 mM and 50 mMsodium phosphate, between 100 and 200 mM sodium chloride, between 5 mMand 25 mM L-Methionine, between 2.5% and 10% Sucrose, between 0.01% and0.1% Tween 20 and between 0.1% and 0.2% (w/v) aluminium hydroxide. Morepreferably, the formulation comprises 10 mM sodium phosphate, 150 mMsodium chloride, 10 mM L-Methionine, 5% Sucrose, 0.05% Tween 20 and0.15% (w/v) aluminium hydroxide at pH 6.7±0.2. Even more preferably, theformulation comprises at least one, at least two, at least three mutantOspA heterodimers according to the invention.

In one embodiment, the pharmaceutical composition comprises 3heterodimers, preferably Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S4D1-S3D1(SEQ ID NO: 194) and Lip-S5D1-S6D1 (SEQ ID NO: 190). Preferably, thethree heterodimers are mixed at a molar ratio of 1:2:1, 1:3:1, 1:1:2,1:1:3, 1:2:2, 1:2:3, 1:3:2, 1:3:3, 2:1:1, 2:1:2, 2:1:3, 2:2:3, 2:2:1,2:3:1, 2:3:2, 2:3:3, 3:1:1, 3:1:2, 3:1:3, 3:2:1, 3:2:2, 3:2:3, 3:3:1,3:3:2, most preferably 1:1:1.

In a further embodiment, the pharmaceutical composition comprises twoheterodimers, preferably Lip-S1 D1-S2D1 (SEQ ID NO: 186) andLip-S5D1-S6D1 (SEQ ID NO: 190), Lip-S1 D1-S2D1 (SEQ ID NO: 186) andLip-S4D1-S3D1 (SEQ ID NO: 194) or Lip-S4D1-S3D1 (SEQ ID NO: 194) andLip-S5D1-S6D1 (SEQ ID NO: 190) in a molar ratio of 1:2, 1:3, 2:1, 3:1,2:3, 3:2, preferably 1:1.

In one embodiment the pharmaceutical composition or vaccine of theinvention further comprises at least one additional antigen (hereinreferred to generically as “combination vaccine”). In a preferredembodiment, the at least one additional antigen is derived from aBorrelia species causing Lyme borreliosis. In various aspects, the atleast one additional antigen is derived from another pathogen,preferably a tick-borne pathogen. In a further aspect, the pathogencauses Rocky Mountain spotted fever, Human granulocytic ehrlichiosis(HGE), Sennetsu Fever, Human Monocytic Ehrlichiosis (HME), Anaplasmosis,Boutonneuse fever, Rickettsia parkeri Rickettsiosis, SouthernTick-Associated Rash Illness (START), Helvetica Spotted fever, 364DRickettsiosis, African spotted fever, Relapsing fever, Tularemia,Colorado tick fever, Tick-borne encephalitis (TBE, also known as FSME),Crimean-Congo hemorrhagic fever, Q fever, Omsk hemorrhagic fever,Kyasanur forest disease, Powassan encephalitis, Heartland virus diseaseor Babesiosis. In a further aspect, the disease is Japaneseencephalitis.

In a further embodiment, the at least one additional antigen is derivedfrom a vector-borne, preferably a tick-borne, pathogen selected from thegroup comprising Borrelia hermsii, Borrelia parkeri, Borrelia duttoni,Borrelia miyamotoi, Borrelia turicatae, Rickettsia rickettsii,Rickettsia australis, Rickettsia conori, Rickettsia helvetica,Francisella tularensis, Anaplasma phagocytophilum, Ehrlichia sennetsu,Ehrlichia chaffeensis, Coxiella burnetii and Borrelia lonestari,Tick-borne encephalitis virus (TBEV aka FSME virus), Colorado tick fevervirus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHFV), OmskHemorrhagic Fever virus (OHFV), Japanese encepalitis virus (JEV) andBabesia spp.

In another aspect, a combination vaccine of the invention comprises anyvaccine composition discussed herein in combination with at least asecond vaccine composition. In some aspects, the second vaccinecomposition protects against a vector-borne disease, preferably atick-borne disease. In various aspects, the second vaccine compositionhas a seasonal immunization schedule compatible with immunizationagainst Borrelia infection or Lyme borreliosis. In other aspects,combination vaccines are useful in the prevention of multiple diseasesfor use in geographical locations where these diseases are prevalent.

In one aspect, the second vaccine composition is a vaccine selected fromthe group consisting of a tick-borne encephalitis vaccine, a Japaneseencephalitis vaccine, and a Rocky Mountain Spotted Fever vaccine. In apreferred aspect, the vaccine composition is FSME-IMMUN® (Baxter),Encepur® (Novartis Vaccines), EnceVir® (Microgen NPO) or TBE MoscowVaccine® (Chumakov Institute of Poliomyelitis and Viral Encephalitidesof Russian Academy of Medical Sciences). In another preferred aspect,the vaccine composition is IXIARO®/JESPECT® (Valneva SE), JEEV®(Biological E, Ltd.) or IMOJEV® (Sanofi Pasteur).

There is further provided a vaccine comprising the pharmaceuticalcomposition, this vaccine may further comprise a pharmaceuticallyacceptable excipient. In a preferred embodiment, the excipient isL-methionine.

The invention also includes immunogenic compositions. In some aspects,an immunogenic composition of the invention comprises any of thecompositions discussed herein and a pharmaceutically acceptable carrier.In various aspects, the immunogenic composition has the property ofinducing production of an antibody that specifically binds an outersurface protein A (OspA) protein. In certain aspects, the immunogeniccomposition has the property of inducing production of an antibody thatspecifically binds Borrelia. In particular aspects, the immunogeniccomposition has the property of inducing production of an antibody thatneutralizes Borrelia. In some aspects, the antibody is produced by ananimal. In further aspects, the animal is a mammal. In even furtheraspects, the mammal is human.

The vaccine preparations containing pharmaceutical compositions of thepresent invention may be used to protect a mammal susceptible toBorrelia infection or treat a mammal with a Borrelia infection, by meansof administering said vaccine via a systemic or mucosal route. Theseadministrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory or genitourinarytracts. Although the vaccine of the invention may be administered as asingle dose, components thereof may also be co-administered together atthe same time or at different times.

In one aspect of the invention is provided a vaccine kit, comprising avial containing a pharmaceutical composition of the invention,optionally in lyophilised form, and further comprising a vial containingan adjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition. In a further aspect, the pharmaceuticalcomposition of the invention may be pre-mixed in a vial, preferably in asyringe.

A further aspect of the invention is a method of preventing or treatingBorrelia infection comprising administering to the host animmunoprotective dose of the pharmaceutical composition or vaccine orkit of the invention. In one embodiment there is provided a method ofpreventing or treating primary and/or recurrence episodes of Borreliainfection comprising administering to the host an immunoprotective doseof the pharmaceutical composition or vaccine or kit of the invention.

A further aspect of the invention is a pharmaceutical composition of theinvention for use in the treatment or prevention of Borrelial disease.In one embodiment there is provided a pharmaceutical composition for usein the treatment or prevention of Borrelia infection.

A further aspect of the invention is the use of the pharmaceuticalcomposition or vaccine or kit of the invention in the manufacture of amedicament for the treatment or prevention of Borrelia infection. In oneembodiment there is provided a pharmaceutical composition of theinvention for use in the manufacture of a medicament for the treatmentor prevention of Borrelia infection.

The invention also includes methods for inducing an immunologicalresponse in a subject. In various aspects, such methods comprise thestep of administering any of the immunogenic compositions or vaccinecompositions discussed herein to the subject in an amount effective toinduce an immunological response. In certain aspects, the immunologicalresponse comprises production of an anti-OspA antibody.

The invention includes methods for preventing or treating a Borreliainfection or Lyme boreliosis in a subject. In various aspects, suchmethods comprise the step of administering any of the vaccinecompositions discussed herein or any of the combination vaccinesdiscussed herein to the subject in an amount effective to prevent ortreat the Borrelia infection or Lyme borreliosis.

The invention includes uses of polypeptides, nucleic acids, antibodies,pharmaceutical compositions or vaccines of the invention for thepreparation of medicaments. Other related aspects are also provided inthe instant invention.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance. The term “comprises” means “includes”. Thus, unless thecontext requires otherwise, the word “comprises”, and variations such as“comprise” and “comprising” will be understood to imply the inclusion ofa stated compound or composition (e.g., nucleic acid, polypeptide,antibody) or step, or group of compounds or steps, but not to theexclusion of any other compounds, composition, steps, or groups thereof.The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example”.

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “pharmaceuticalcompositions” of the invention, and vice versa.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewof al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The term “plurality” refers to two or more. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Additionally,numerical limitations given with respect to concentrations or levels ofa substance, such as an antigen, may be approximate.

A preferable carrier or excipient for the polypeptides according to thepresent invention in their diverse embodiments, or a nucleic acidmolecule according to the present invention is an immunostimulatorycompound such as an adjuvant for further stimulating the immune responseto the polypeptide according to the present invention or a codingnucleic acid molecule thereof.

Adjuvants which may be used in compositions of the invention include,but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.,oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates),sulphates, etc., or mixtures of different mineral compounds, with thecompounds taking any suitable form (e.g., gel, crystalline, amorphous,etc.), and with adsorption being preferred. The mineral containingcompositions may also be formulated as a particle of metal salt.

A useful aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92. Anotheruseful aluminium-based adjuvant is AS04, a combination of aluminiumhydroxide+monophosphoryl lipid A (MPL).

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-in-water emulsions, such as MF59 (5% Squalene, 0.5%Tween 80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer), AS03 (squalene, DL-α-tocopherol and Tween 80) and AF03(squalene, Montane® 80 and Eumulgon® B1 PH). Complete Freund's adjuvant(CFA) and incomplete Freund's adjuvant (1FA) may also be used.

Useful oil-in-water emulsions typically include at least one oil and atleast one surfactant, with the oil(s) and surfactant(s) beingbiodegradable (metabolizable) and biocompatible. The oil droplets in theemulsion are generally less than 1 μm in diameter, with these smallsizes being achieved with a microfluidizer to provide stable emulsions.Droplets with a size less than 220 nm are preferred as they can besubjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and gains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g., obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the gain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Other preferred oils are thetocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWF AXr™ tradename, such as linear EO/PO blockcopolymers; octoxynols, which can vary in the number of repeating ethoxy(oxy-12-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy) polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g., Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X 100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Preferably, substantially all (e.g. at least 90% by number) of the oildroplets have a diameter of less than μm, e.g. <750 nm, <500 nm, <400nm, <300 nm, <250 nm, <220 nm, <200 nm, or smaller. One specific usefulsubmicron emulsion consists of squalene, Tween 80, and Span 85. Thecomposition of the emulsion by volume can be about 5% squalene, about0.5% polysorbate 80 and about 0.5°/s Span 85. In weight terms, theseratios become 4.3% squalene. 0.5% polysorbate 80 and 0.48% Span 85. TheMF59 emulsion advantageously includes citrate ions e.g. 10 mM sodiumcitrate buffer.

C. Saponin Formulations

Saponin formulations may also be used as adjuvants in the invention.Suponins are a heterogeneous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree has been widely studied as adjuvant.Saponin can also be commercially obtained from Smilax ornata(sarsaparilla), Gypsophilla paniculata (bridal veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. Saponin formulations may also comprise a sterol, suchas cholesterol.

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of QS7, QS 17, QS 18, QS21,QH-A, QH-B and QH-C. Optionally, the ISCOMS may be devoid of additionaldetergent.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retroviruses, Norwalk virus, Human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pi).

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. Such “smallparticles” of 3dMPL are small enough to be sterile filtered through a0.22 μm membrane. Other non-toxic LPS derivatives include monophosphoryllipid A mimics, such as aminoalkyl glucosaminide phosphate derivativese.g. RC-529 and the synthetic phospholipid dimer, E6020.

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. Immunostimulatory oligonucleotides suitable for use asadjuvants in the invention include nucleotide sequences containing a CpGmotif (a dinucleotide sequence containing an unmethylated cytosinelinked by a phosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. The CpG sequence may be directed to TLR9, such as themotif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing aTh1 immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. Preferably, the CpG is aCpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. Aparticularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC31®. Thus an adjuvant used with theinvention may comprise a mixture of (i) an oligonucleotide (e.g. between15-40 nucleotides) including at least one (and preferably multiple) Cplmotifs (i.e. a cytosine linked to an inosine to form a dinucleotide),and (ii) a polycationic polymer, such as an oligopeptide (e.g. between5-20 amino acids) including at least one (and preferably multiple)Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be adeoxynucleotide comprising the 26-mer sequence 5′-(dIdC)₁₃-3′ (SEQ IDNO: 32). The polycationic polymer may be a peptide comprising the 11-meramino acid sequence KLKLLLLLKLK (SEQ ID NO: 33).

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide,which has the amino acid sequenceNH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO: 31). Relatedor derived cathelin substances contain the whole or parts of thecathelin sequence with at least 15-20 amino acid residues. Derivationsmay include the substitution or modification of the natural amino acidsby amino acids which are not among the 20 standard amino acids.Moreover, further cationic residues may be introduced into such cathelinmolecules. These cathelin molecules are preferred to be combined withthe antigen. These cathelin molecules surprisingly have turned out to bealso effective as an adjuvant for an antigen without the addition offurther adjuvants. It is therefore possible to use such cathelinmolecules as efficient adjuvants in vaccine formulations with or withoutfurther immune activating substances.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), Vibriocholera (Cholera toxin “CT”), or Bordetella pertussis (Pertussis toxin“PT”). The use of detoxified ADP-ribosylating toxins as mucosaladjuvants and as parenteral adjuvants is known. The toxin or toxoid ispreferably in the form of a holotoxin, comprising both A and B subunits.Preferably, the A subunit contains a detoxifying mutation; preferablythe B subunit is not mutated. Preferably, the adjuvant is a detoxifiedLT mutant such as LT-K63, LT-R72, LT-G192 or dmLT. A useful CT mutant isCT-E29H.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophagecolony stimulating factor and tumor necrosis factor. A preferredimmunomodulator is IL-12.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres or mucoadhesives such as cross-linked derivatives ofpolyacrylic acid, polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention.

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g., a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, apoly(lactide-co-glycolide) etc.), wherein poly(lactide-co-glycolide) arepreferred, optionally treated to have a negatively-charged surface (e.g.with SDS) or a positively-charged surface (e.g., with a cationicdetergent, such as CTAB).

I. Liposomes

Examples of liposome formulations suitable for use as adjuvants areknown.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. Such formulations further includepolyoxyethylene sorbitan ester surfactants in combination with anoctoxynol as well as polyoxyethylene alkyl ethers or ester surfactantsin combination with at least one additional non-ionic surfactant such asan octoxynol. Preferred polyoxyethylene ethers are selected from thefollowing group: polyoxyethylene-9-lauryl ether (laureth 9),polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

K. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-5n-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

L. Imidazoquinolone Compounds.

Examples of imidazoquinoline compounds suitable for use as adjuvants inthe invention include Imiquimod and its homologues (e.g., “Resiquimod3M”).

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above.

Preferably, the immunostimulatory compound in the pharmaceuticalpreparation according to the present invention is selected from thegroup of polycationic substances, especially polycationic peptides,immunostimulatory nucleic acids molecules, preferably immunostimulatorydeoxynucleotides, oil-in-water or water-in-oil emulsions, MF59,aluminium salts, Freund's complete adjuvant, Freund's incompleteadjuvant, neuroactive compounds, especially human growth hormone, orcombinations thereof.

The use of an aluminium hydroxide and/or aluminium phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts.

Also, the pharmaceutical composition in accordance with the presentinvention is a pharmaceutical composition which comprises at least anyof the following compounds or combinations thereof: the nucleic acidmolecules according to the present invention, the polypeptides accordingto the present invention in their diverse embodiments, the vectoraccording to the present invention, the cells according to the presentinvention and the antibody according to the present invention. Inconnection therewith, any of these compounds may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such carriers may include, butare not limited to, saline, buffered saline, dextrose, water, glycerol,ethanol and combinations thereof. The formulation should suit the modeof administration.

In one embodiment, the pharmaceutical composition comprises astabilizer. The term “stabilizer” refers to a substance or vaccineexcipient which protects the immunogenic composition of the vaccine fromadverse conditions, such as those which occur during heating orfreezing, and/or prolongs the stability or shelf-life of the immunogeniccomposition in a stable and immunogenic condition or state. Examples ofstabilizers include, but are not limited to, sugars, such as sucrose,lactose and mannose; sugar alcohols, such as manitol; amino acids, suchas glycine or glutamic acid; and proteins, such as human serum albuminor gelatin.

The pharmaceutical compositions of the present invention may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intratrachealor intradermal routes, among others. In a preferred embodiment, thepharmaceutical compositions are administered subcutaneously orintramuscularly, most preferably intramuscularly.

In therapy or as a prophylactic, the active agent of the pharmaceuticalcomposition of the present invention may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition, preferably the pharmaceutical compositionmay be formulated for topical application, for example in the form ofointments, creams, lotions, eye ointments, eye drops, ear drops,mouthwash, impregnated dressings and sutures and aerosols, and maycontain appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. Such topical formulations may also containcompatible conventional carriers, for example cream or ointment bases,and ethanol or oleyl alcohol for lotions. Such carriers may constitutefrom about 1% to about 98% by weight of the formulation; more usuallythey will constitute up to about 80% by weight of the formulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

In a preferred embodiment the pharmaceutical composition is a vaccinecomposition. Preferably, such vaccine composition is conveniently ininjectable form. Conventional adjuvants may be employed to enhance theimmune response. A suitable unit dose for vaccination with a proteinantigen is for adults between 0.02 μg and 3 μg antigen per kg bodyweight and for children between 0.2 μg and 10 μg antigen per kg bodyweight, and such dose is preferably administered 1 to 3 times atintervals of 2 to 24 weeks.

At the indicated dose range, no adverse toxicological effects areexpected with the compounds of the invention, which would preclude theiradministration to suitable individuals.

As an additional aspect, the invention includes kits which comprise oneor more pharmaceutical formulations for administration to a subjectpackaged in a manner which facilitates their use for administration tosubjects, in a preferred embodiment, the kits comprise the formulationin a final volume of 2 mL, more preferably in a final volume of 1 mL.

In a specific embodiment, the invention includes kits for producing asingle dose administration unit. The kits, in various aspects, eachcontain both a first container having a dried protein and a secondcontainer having an aqueous formulation. Also included within the scopeof this invention are kits containing single and multi-chamberedpre-filled syringes (e.g., liquid syringes and lyosyringes).

In another embodiment, such a kit includes pharmaceutical formulationdescribed herein (e.g., a composition comprising a therapeutic proteinor peptide), packaged in a container such as a sealed bottle or vessel,with a label affixed to the container or included in the package thatdescribes use of the compound or composition in practicing the method.In one embodiment, the pharmaceutical formulation is packaged in thecontainer such that the amount of headspace in the container (e.g., theamount of air between the liquid formulation and the top of thecontainer) is very small. Preferably, the amount of headspace isnegligible (i.e., almost none).

In one aspect, the kit contains a first container having a therapeuticprotein or peptide composition and a second container having aphysiologically acceptable reconstitution solution for the composition,in one aspect, the pharmaceutical formulation is packaged in a unitdosage form. The kit optionally further includes a device suitable foradministering the pharmaceutical formulation according to a specificroute of administration. In some aspects, the kit contains a label thatdescribes use of the pharmaceutical formulations.

The pharmaceutical composition can contain a range of differentantigens. Examples of antigens are whole-killed or attenuated organisms,subfractions of these organisms, proteins, or, in their most simpleform, peptides. Antigens can also be recognized by the immune system inthe form of glycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used, since cytotoxicT-cells (cm) recognize antigens in the form of short, usually 8-11 aminoacids long, peptides in conjunction with major histocompatibilitycomplex (MHC). B cells can recognize linear epitopes as short as 4 to 5amino acids, as well as three-dimensional structures (conformationalepitopes).

In a preferred embodiment, the pharmaceutical composition of the thirdaspect additionally comprises a hyperimmune serum-reactive antigenagainst a Borrelia protein or an active fragment or variant thereof,such as, e.g., the antigens, fragments and variants as described in WO2008/031133.

According to the invention, the pharmaceutical composition according tothe third aspect may be used as a medicament, particularly as a vaccine,particularly in connection with particularly a disease or diseasedcondition which is caused by, linked or associated with Borrelia.

The pharmaceutical composition of the present invention may be used as amedicament, particularly as a vaccine, particularly in connection with adisease or disease condition which is caused by, linked with orassociated with Borrelia, more preferably any pathogenic Borreliaspecies and more preferably in a method for treating or preventing aBorrelia infection, particularly a B. burgdorferi s.s., B. garinii, B.afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B.lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi or B.sinica infection, preferably a B. burgdorferi s.s., B. afzelii or B.garinii infection.

In connection therewith, it should be noted that the various Borreliaspecies, including B. burgdorferi s.l., comprise several species andstrains including those disclosed herein. A disease related, caused orassociated with the bacterial infection to be prevented and/or treatedaccording to the present invention includes Lyme borreliosis (Lymedisease). Further aspects, symptoms, stages and subgroups of Lymeborreliosis as well as specific groups of patients suffering from suchdisease as also disclosed herein, including in the introductory part,are incorporated herein by reference. More specifically, Lymeborreliosis generally occurs in stages, with remission and exacerbationswith different clinical manifestation at each stage. Early infectionstage 1 consists of localized infection of the skin, followed withindays or weeks by stage 2, disseminated infection, and months to yearslater by stage 3, persistent infection. However, the infection isvariable; some patients have only localized infections of the skin,while others display only later manifestations of the illness, such asarthritis.

In a fourth aspect, the present invention relates to a method oftreating or preventing a Borrelia infection in a subject in needthereof, comprising the step of administering to the subject atherapeutically effective amount of a pharmaceutical compositionaccording to the third aspect.

The term “subject” is used throughout the specification to describe ananimal, preferably a mammal, more preferably a human, to whom atreatment or a method according to the present invention is provided.For treatment of those infections, conditions or disease states whichare specific for a specific animal such as a human patient, the termpatient refers to that specific animal. Preferably, the subject is ahuman; however, the medical use of the composition may also includeanimals such as poultry including chicken, turkey, duck or goose,livestock such as horse, cow or sheep, or companion animals such as dogsor cats.

The term “effective amount” is used throughout the specification todescribe an amount of the present pharmaceutical composition which maybe used to induce an intended result when used in the method of thepresent invention. In numerous aspects of the present invention, theterm effective amount is used in conjunction with the treatment orprevention. In other aspects, the term effective amount simply refers toan amount of an agent which produces a result which is seen as beingbeneficial or useful, including in methods according to the presentinvention where the treatment or prevention of a Borrelia infection issought.

The term effective amount with respect to the presently describedcompounds and compositions is used throughout the specification todescribe that amount of the compound according to the present inventionwhich is administered to a mammalian patient, especially including ahuman patient, suffering from a Borrelia-associated disease, to reduceor inhibit a Borrelia infection.

In a preferred embodiment, the method of immunizing a subject accordingto the fourth aspect comprises the step of administering to the subjecta therapeutically effective amount of a pharmaceutical composition ofthe third aspect of the current invention.

The method comprises inducing an immunological response in an individualthrough gene therapy or otherwise, by administering a polypeptide ornucleic acid according to the present invention in viii in order tostimulate an immunological response to produce antibodies or acell-mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether ornot that disease is already established within the individual.

The products of the present invention, particularly the polypeptides andnucleic acids, are preferably provided in isolated form, and may bepurified to homogeneity. The term “isolated” as used herein meansseparated “by the hand of man” from its natural state; i.e., if itoccurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a naturally-occurring nucleic acidmolecule or a polypeptide naturally present in a living organism in itsnatural state is not “isolated”, but the same nucleic acid molecule orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. As part of or followingisolation, such nucleic acid molecules can be joined to other nucleicacid molecules, such as DNA molecules, for mutagenesis, to form fusiongenes, and for propagation or expression in a host, for instance. Theisolated nucleic acid molecules, alone or joined to other nucleic acidmolecules such as vectors, can be introduced into host cells, in cultureor in whole organisms. Introduced into host cells in culture or in wholeorganisms, such DNA molecules still would be isolated, as the term isused herein, because they would not be in their naturally-occurring formor environment. Similarly, the nucleic acid molecules and polypeptidesmay occur in a composition, such as medium formulations, solutions forintroduction of nucleic acid molecules or polypeptides, for example,into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated nucleic acid molecules or polypeptideswithin the meaning of that term as it is employed herein.

The invention is not limited to the particular methodology, protocolsand reagents described herein because they may vary. Furthermore, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention. As used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. Similarly, the words “comprise”. “contain” and“encompass” are to be interpreted inclusively rather than exclusively.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, and materials are described herein.

The present invention is further illustrated by the following Figures,Tables, Examples and the Sequence listing, from which further features,embodiments and advantages may be taken. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to the person skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is thus to beunderstood that such equivalent embodiments are to be included herein.

In connection with the present invention

FIG. 1 shows the amino acid alignment of OspA serotypes 1-6 fromBorrelia.

FIG. 2 schematically shows the production of mutant OspA fragmentheterodimers according to the current invention.

FIG. 3 schematically represents the polypeptide components of onepossible pharmaceutical composition of the current invention comprisingthree different mutant OspA heterodimers, a “combination vaccine”.

FIG. 4 shows the chemical structure of Pam₃Cys, an example of a fattyacid substituted cysteine, such as would be found at the N-terminus oflipidated polypeptides of the current invention.

FIG. 5 shows the binding of antibodies from mice immunized with mutantOspA fragment heterodimer polypeptides of the invention to the cellsurface of Borrelia of OspA serotypes 1-6.

Table 1 shows the thermal stability of the folding of mutant serotype 2OspA fragments with disulfide bond types from D1 to D5 (fornomenclature, see Table A-4) compared to the wild-type serotype 2 OspAfragment without disulfide bonds (DO).

Table 2 shows the protection of mice from B. afzelii (strain IS1)infection by the Tick Challenge Method following immunization withmutant serotype 2 OspA fragments with disulfide bond types D1 to D5 (fornomenclature, see Table A-4), including control groups of mice immunizedwith PBS, full-length OspA or the wild-type serotype 2 OspA fragment(S2D0-His).

Table 3 shows the protection of mice from B. afzelii (strain IS)infection by the Tick Challenge Method following immunization withlipidated mutant serotype 2 OspA fragments with disulfide bond types D1,D3 and D4 (Lip-S2D1-His, Lip-S203-His and Lip-S2D4-His), includingcontrol groups of mice immunized with PBS or full-length OspA protein.

Table 4 shows the protective capacity of mutant OspA heterodimers of theinvention in in vivo Borrelia challenge models. Mice were immunized withLip-S1D1-S2D1-His, Lip-S4D1-S3D1-His, Lip-S4D1-S3D1 or Lip-S5D1-S6D1-Hisand challenged with the indicated Borrelia OspA serotype via Tick orNeedle Challenge Method, as indicated. The control group in eachexperiment was immunized with Al(OH)3 adjuvant alone.

Table 5 shows the protective capacity of the combination vaccine of theinvention against challenge in vivo with OspA serotype 1 Borrelia(strain N40 in needle challenge method) and OspA serotype 2 Borrelia(strain IS1 in the tick challenge method). Mice were immunized with thethree antigens Lip-S1 D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-56D1 togetherin a 1:1:1 ratio (combination vaccine) or with the indicated controlantigens and challenged with Borrelia via Tick or Needle ChallengeMethod, as indicated. The control group in each experiment was immunizedwith Al(OH)3 adjuvant alone.

The figures and tables which may be referred to in the specification aredescribed below in more detail.

FIG. 1 Amino acid sequence alignment of OspA serotypes one through six(ST1-ST6), excluding the N-terminal lipidation signal sequence (aminoacids 1-16), which is truncated during processing. The alignmentillustrates that the membrane-associated N-terminal portion of theprotein has a more highly-conserved amino acid sequence than the moreexposed C-terminal portion. The full-length OspA sequences (excludingthe N-terminal lipidation signal sequences, amino acids 1-16, which arenot shown in the figure) are as follows: OspA ST3, SEQ ID NO: 21: OspAST1, SEQ ID NO: 20; OspA ST4, SEQ ID NO: 22; OspA ST5, SEQ ID NO: 23;OspA ST6, SEQ ID NO: 24; OspA ST2, SEQ ID NO: 19.

FIG. 2 Production of a mutant OspA heterodimer of the inventioncomprising mutant OspA C-terminal fragments from two different OspAserotypes of Borrelia sp. (A) Schematic representation of a nucleic acidencoding a lipidated mutant OspA heterodimer. The components, from 5′ to3′, comprise the coding sequences for a lipidation signal sequence (Lipsignal), a small cysteine-containing peptide for N-terminal lipidation(Lipidation peptide=LP), a mutant C-terminal fragment of OspA with twonon-native cysteines, a short linker peptide (LN1), followed by a secondmutant OspA C-terminal fragment with two non-native cysteines. (B) Theintermediate mutant OspA heterodimer polypeptide comprises the nascentproduct directly following translation of the nucleic acid construct.From the N- to the C-terminus, this polypeptide consists of a lipidationsignal sequence (Lip signal), a cysteine-containing peptide forlipidation (LP), a mutant OspA fragment with a non native disulfidebond, a short linker peptide (LN1), followed by a second mutant OspAfragment with a non native disulfide bond. (C) The final lipidatedmutant OspA heterodimer polypeptide after post-translationalmodification. The heterodimer, from the N- to the C-terminus, consistsof a short cysteine-containing peptide with the N-terminal cysteinelipidated (indicated by “Lip”), a mutant OspA fragment stabilized by adisulfide bond, a linker peptide (LN1), and a second mutant OspAfragment stabilized by a disulfide bond. The lipidation signal sequenceis cleaved off during post-translational modification of the polypeptideas shown.

FIG. 3 An example of a preferred pharmaceutical composition according tothe current invention. Three mutant OspA heterodimers, each comprisingmutated OspA fragments from two different Borrelia OspA serotypes arepresent in the composition, together providing OspA antigens from sixdifferent Borrelia OspA serotypes. Such a pharmaceutical compositionenables simultaneous immunization against six Borrelia serotypes.

FIG. 4 Illustration of the chemical structure of Pam₃Cys, an example ofa fatty acid substitution of the N-terminal cysteine of full-lengthwild-type OspA protein as well as of lipidated mutant OspA fragmentmonomers and heterodimers of the invention. During post-translationalmodification of a full-length OspA protein or polypeptides of theinvention, the N-terminal lipidation signal sequence is cleaved off andfatty acids, most commonly three palmitoyl moieties (“Pam₃”), areenzymatically covalently attached to the N-terminal cysteine residue(the sulfur atom, “S”, is indicated by an arrow). The remaining residuesof the polypeptide chain, which are located C-terminally from thePam₃Cys residue, are represented by “Xn”. (Modified from Bouchon, et al.(1997) Analytical Biochemistry 246: 52-61.)

FIG. 5 Binding of antibodies from immunized mice to the cell surface ofBorrelia spirochetes. Mice were immunized three times with 1 μg each ofthe indicated antigens: Lipidated and His-tagged full-length OspAproteins of OspA serotypes 1-6; Lip-S1 D1-S2D1, Lip-S4D1-S3D1 orLip-S5D1-S6D1 alone, or Lip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1together in a 1:1:1 ratio (“combination vaccine”) at two week intervalsand sera were collected at one week after the last dose. Severaldilutions of the sera were tested for binding to the cell surface ofBorrelia via cell staining and flow cytometry. Fluorescent intensityvalues observed when staining with sera collected from control miceimmunized with Al(OH)₃ adjuvant alone were subtracted to account fornon-specific binding. (Borrelia used were: B. burgdorferi. OspA serotype1, strain N40; B. afzelii, OspA serotype 2, strain “C”; B. garinii, OspAserotype 3, strain “D”; B. bavariensis, OspA serotype 4, strain Fin; B.garinii, OspA serotype 5, strain “E”; B. garinii, OspA serotype 6,strain “B”.)

TABLE 1 Thermal stability of non-lipidated, His-tagged B. afzelii K78mutant serotype 2 OspA fragments with different placement of disulfidebonds. Mutant serotype 2 OspA fragments with different cysteine bondtypes (see Table A-4) were solubilized in 50 mM Tris-HCl, 150 mM NaCl(pH 8.0) and tested for thermal stability compared with the wild-typeserotype 2 OspA fragment (S2D0). The presence of a disulfide bondresulted in an increased melting temperature compared to the wild-typeserotype 2 OspA fragment. Serotype 2 OspA Melting mutant fragment SEQ IDNO: temperature (° C.) S2D0-His* 1 47.6 S2D1-His 2 70.4 S2D2-His 3 54.6S2D3-His 4 58.6 S2D4-His 5 58.4 S2D5-His 6 53.8 *see Tables A-4 and A-5for nomenclature.

TABLE 2 Protective capacity of decreasing doses of non-lipidatedHis-tagged mutant serotype 2 OspA fragments against B. afzelii (serotype2) infection by the Tick Challenge Method. Five non-lipidated His-taggedmutant serotype 2 OspA fragments were tested for protective capacity attwo different doses (30 μg and 5 μg) and compared with the wild-typeserotype 2 OspA fragment. Groups of mice immunized with Al(OH)₃ adjuvantalone or with non-lipidated full-length serotype 2 OspA served asnegative and positive controls, respectively. All antigens wereHis-tagged and non-lipidated. The data presented combine the results ofseveral experiments performed under identical conditions. Tick challenge3 × 30 μg 3 × 5 μg (OspA serotype 2: B. (11 experiments) (4 experiments)Immunogen afzelii, strain IS1) Infected/total Infected/total Al(OH)₃adjuvant alone Tick (OspA-ST2) 58/62  20/23  Full-length OspA K78-HisTick (OspA-ST2) 1/72 1/25 (SEQ ID NO: 209) S2D0-His Tick (OspA-ST2)15/20  8/16 (SEQ ID NO: 1) S2D1-His Tick (OspA-ST2) 1/26 1/25 (SEQ IDNO: 2) S2D2-His Tick (OspA-ST2) 0/26 4/26 (SEQ ID NO: 3) S2D3-His Tick(OspA-ST2) 0/34 1/21 (SEQ ID NO: 4) S2D4-His Tick (OspA-ST2) 2/30 4/27(SEQ ID NO: 5) S2D5-His Tick (OspA-ST2) 5/35 2/11 (SEQ ID NO: 6)

TABLE 3 Protective capacity of decreasing doses of lipidated His-taggedmutant serotype 2 OspA fragments against B. afzelii infection by theTick Challenge Metbod. Three lipidated His-tagged mutant scrotype 2 OspAfragments with different disulfide bond types were tested for protectivecapacity at three different doses (3.0 μg, 1.0 μg and 0.3 μg). Groups ofmice immunized with Al(OH)₃ adjuvant alone or with non-lipidatedfull-length serotype 2 OspA served as negative and positive controls,respectively. The data presented combine the results of severalexperiments performed under identical conditions. Tick challenge 3 × 3.0μg 3 × 1.0 μg 3 × 0.3 mg (OspA serotype 2: B. (5 experiments) (5experiments) (4 experiments) Immunogen afzelii, strain IS1)Infected/total Infected/total Infected/total Al(OH)₃ adjuvant alone Tick(OspA-ST2) 58/59  — — (control for all doses) Full-length OspA K78-HisTick (OspA-ST2) 0/14 0/21 1/20 (SEQ ID NO: 209) Lip-S2D1-His Tick(OspA-ST2) 0/17 5/31 1/29 (SEQ ID NO: 141) Lip-S2D3-His Tick (OspA-ST2)1/15 1/12 5/19 (SEQ ID NO: 143) Lip-S2D4-His Tick (OspA-ST2) 0/8  0/250/34 (SEQ ID NO: 144)

TABLE 4 Protective capacity of mutant OspA heterodimers of the inventionagainst in vivo Borrelia challenge via Needle or Tick Challenge Methods.Groups of mice were immunized three times at two week intervals with theindicated doses of OspA heterodimer or Al(OH)₃ adjuvant alone.Immunogens used were Lip-S1D1-S2D1-His (challenged with BorreliaOspA-ST1, Experiments 1-3), Lip-S1D1-S2D1-His, Lip-S4D1-S3D1-His andLip-SSD1-S6D1-His, separately (challenged with Borrelia OspA-ST2,Experiments 4-6), Lip-S4D1-S3D1 (challenged with Borrelia OspA-ST4,Experiments 7 and 8) and Lip-S5D1-S6D1-His (challenged with BorreliaOspA-ST5, Experiments 9 and 10; challenged with Borrelia OspA-ST6,Experiments 11 and 12). Immunized mice were challenged two weeks afterthe last immunization via Tick or Needle Challenge Models as indicated.Needle challenge (OspA-serotype 1: B. Infected/Total Immunogen Doseburgdorferi s.s., strain N40) Exp. 1 Exp. 2 Exp. 3 Lip-S1D1-S2D1-His 3 ×5.0 μg Needle (OspA-ST1)   0/10***  0/9*** 4/10** (SEQ ID NO: 49)Al(OH)₃ adjuvant alone — Needle (OspA-ST1) 10/10  8/10 10/10   Tickchallenge (OspA-serotype 2: B. Immunogen Dose afzelii, strain IS1) Exp.4 Exp. 5 Exp. 6 Lip-S1D1-S2D1-His 3 × 2.0 μg Tick (OspA-ST2)   0/10*** 0/9*** 0/6*** (SEQ ID NO: 49) Lip-S4D1-S3D1-His 3 × 2.0 μg Tick(OspA-ST2)  0/9*** 2/7* 0/6*** (SEQ ID NO: 81) Lip-S5D1-S6D1-His 3 × 2.0μg Tick (OspA-ST2)  0/7***  0/9*** 0/6*** (SEQ ID NO: 65) Al(OH)₃adjuvant alone — Tick (OspA-ST2) 9/9  8/8  7/7   Needle challenge(OspA-serotype 4. B. Immunogen Dose bavariensis, strain Scf) Exp. 7 Exp.8 Lip-S4D1-S3D1 3 × 5.0 μg Needle (OspA-ST4)  2/10**   1/10*** — (Seq IDNo: 194) Al(OH)₃ adjuvant alone — Needle (OspA-ST4) 9/10 9/10 — Needlechallenge (OspA-serotype 5: B. Immunogen Dose garinii) Exp. 9 Exp. 10Lip-SSDI-S6DI-His 3 × 5.0 μg Needle (OspA-ST5) 1/10 2/10 — (SEQ ID NO:65) Al(OH)₃ adjuvant alone — Needle (ST5) 6/10 6/10 — Needle challenge(OspA-serotype 6: B. Immunogen Dose garinii) Exp. 11 Exp. 12Lip-S5D1-S6D1-His 3 × 5.0 μg Needle (OspA-ST6)  2/10**   2/10*** — (SEQID NO: 65) Al(OH)₃ adjuvant alone — Needle (OspA-ST6) 9/10 10/10  —P-value; Fisher's exact test, two tailed. *significant (<0.05), **highlysignificant (<0.01), ***extremely significant (<0.001)

TABLE 5 Protective capacity of the mutant OspA beterodimer combinationvaccine of the invention against OspA serotype 1 and serotype 2 Borreliachallenge. Groups of mice were immunized three times with the indicateddoses of immunogen or Al(OH)₃ adjuvant alone at two-week intervals.Immunogens used were a 1:1:1 combination of the mutant OspA heterodimersLip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1 (combination vaccine),Lip-S1D1-S2D1, Lip-OspA1-His and Chimeric OspA S1/ST2. Immunized micewere challenged two weeks after the last immunization via the TickChallenge Method (ST2, Experiments 13 and 14) or the Needle ChallengeMethod (ST1, Experiments 15 and 16). Tick challenge (OspA-serotype 2: BInfected/Total Immunogen Dose afzelii, strain IS1) Exp. 13 Exp. 14Lip-S1D1-S2D1 3 × 5.0 μg Tick (OspA-ST2) 0/6***  0/7**  (SEQ ID NO: 186)Combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 186) 3 × 5.0 μg Tick(OspA-ST2) 0/9***  0/6**  Lip-S4D1-S3D1 (Seq ID No: 194) 3 × 5.0 μgLip-S5D1-S6D1 (Seq ID No: 190) 3 × 5.0 μg Al(OH)₃ adjuvant alone — Tick(OspA-ST2) 7/7   6/7   Needle challenge (OspA-serotype 1: B. burgdorferis.s., Immunogen Dose strain ZS7) Exp. 15 Exp. 16 Lip-S1D1-S2D1 3 × 1.0μg Needle (OspA-ST1) 0/10*** 0/10*** (Seq ID No: 186) Lip-OspA1-His 3 ×1.0 μg Needle (OspA-ST1) 0/10*** 0/10*** (Seq ID No: 210) Chimeric OspAST1/ST2 3 × 1.0 μg Needle (OspA-ST1) 0/10*** 0/10*** (Seq ID No: 212)Combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 186) 3 × 1.0 μg Needle(OspA-ST1) 0/10*** 0/10*** Lip-S4D1-S3D1 (Seq ID No: 194) 3 × 1.0 μgLip-S5D1-S6D1 (Seq ID No: 190) 3 × 1.0 μg Al(OH)₃ adjuvant alone —Needle (OspA-ST1) 10/10   10/10   P-value; Fisher's exact test, twotailed. *significant (<0.05), **highly significant (<0.01), ***extremelysignificant (<0.001)

EXAMPLES Example 1. Assessment of Thermal Stability of Mutant Serotype 2OspA Fragments Experimental Procedures Thermal Stability

The melting temperatures (T_(m)) of non-lipidated mutant serotype 2 OspAfragment monomers were determined by the fluorescence-based thermalshift assay described by Pantoliano, et al. (J. Biomol Screen 6:429-440(2001)). The fluorescent dye SYPRO® Orange protein gel stain (suppliedas a 5000×concentrate in DMSO by Sigma, U.S.A.) was used to monitorprotein unfolding. In each well, 7.5 μL of SYPRO® Orange (diluted 1:1000from the stock solution) and 17.5 μL of a solution of protein (1 μg or 2μg) in buffer were combined. The protein samples were heated from 25° C.to 95′C at a rate of 0.2° C./10 sec in the CFX96 Real-time DetectionSystem (Bio-Rad, LISA) and fluorescent changes were monitored.Fluorescence intensity was measured with excitation and emissionwavelengths of 490 and 575 nm, respectively. The T_(m) was determinedusing the Bio-Rad CFX Manager 2.0 program. The T_(m), values ofnon-lipidated His-tagged serotype 2 OspA mutant fragments were measuredin four different buffer systems: 50 mM Tris-HCl, 150 mM NaCl (pH 9.0);50 mM Tris-HCl, 150 mM NaCl (pH 8.0); PBS (pH 7.4); and 25 mM HEPES, 150mM NaCl (pH 6.5), using the non-lipidated serotype 2 OspA wild-typefragment (S2D0) as a control.

Results

In all cases, mutant serotype 2 OspA fragments with an introducedcysteine bond had higher T_(m) than the wild-type serotype 2 OspAfragment (S2D0) (see Table 1). The T_(m) was tested in four differentbuffer systems with similar results (data for proteins dissolved in 50mM Tris-HCl, 150 mM NaCl (pH 8.0) is shown in Table 1), indicating thatthe stability of the proteins is similar over a wide pH range. Thisresult lends credence to the hypothesis that the introduced disulfidebond stabilizes the OspA fragment.

Example 2. Assessment of the Protective Capacity of Non-LipidatedHis-Tagged Mutant Serotype 2 OspA Fragment Monomers in the TickChallenge Method (ST2, B. afzelii) Experimental Procedures Cloning andExpression of Recombinant Proteins

The the wild-type serotype 2 OspA fragment as well as the mutantserotype 2 OspA fragments with cysteine bond types 1-5 (SEQ ID NOs: 1,2, 3, 4, 5 and 6, respectively), were codon-optimized for E. coliexpression by GenScript, USA. The non-lipidated mutant serotype 2 OspAfragments were C-terminally His-tagged for purification purposes. Genefragments were cloned into the pET28b(+) vector (Novagen, USA), a vectorcontaining a Kanamycin resistance cassette as well as a T7 promoter. Themonomers were expressed in BL21 Star™ (DE3) cells (Invitrogen, USA) at37° C. by the addition of IPTG. Cells were collected after 4 h bycentrifugation and the pellet was stored at 70° C. for up to 12 monthsprior to further processing.

Purification of Non-Lipidated His-Tagged Wild-Type and Mutant OspAFragment Monomer Proteins

Cells were disrupted mechanically by high-pressure homogenization andthe soluble fraction containing the His-tagged OspA fragments wasapplied to a Ni-sepharose column (Ni Sepharose™ 6 Fast Flow: GEHealthcare, United Kingdom) and the His-tagged OspA fragments wereeluted on an Imidazole gradient (0-250 mM). Pooled fractions werefurther purified over a gel filtration column (Superdex 200, GEHealthcare) followed by a buffer exchange column (Sephadex G-25, GEHealthcare). His-tagged OspA fragment peaks were pooled on the basis ofthe analytical size exclusion column and reversed phase chromatography.After sterile filtration, the purified proteins were stored at −20° C.until formulation.

Immunization of Mice

Female C3H/HeN (H-2^(k)) mice were used for all studies (Harlan, Italy).Prior to each challenge, groups of five 8-week-old mice were bled viathe tail vein and pre-immune sera were prepared and pooled. Fivenon-lipidated mutant serotype 2 OspA fragment proteins (S2D1-5, SEQ IDNOs: 2, 3, 4, 5 and 6, respectively), were tested in fifteen separateexperiments. Three subcutaneous (s.c.) immunizations of 100 μL, wereadministered at two week intervals. Doses used were 30 and 5 μg of therespective protein, tested in 11 and 4 experiments respectively. Allformulations included aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. One week after the third immunization, blood wascollected and hyper-immune sera were prepared. In each experiment, onegroup injected with PBS formulated with Al(OH)₃ was included as anegative control and one group of mice was immunized with S2D0, thewild-type C-terminal OspA fragment from B. afzelii strain K78 (SEQ IDNO: 1). Another group immunized with a non-lipidated full-lengthwild-type OspA protein from B. afzelii, strain K78 (SEQ ID NO: 209),also formulated with 0.15% Al(OH)₃, was included as positive control ineach animal study. All animal experiments were conducted in accordancewith Austrian law (BGB1 Nr. 501/1989) and approved by“Magistratsabteilung 58”.

Tick Challenge of Immunized Mice and Collection of Sera and Tissues(Herein Referred to Also as “Tick Challenge Method”)

Tick challenge of immunized mice was done two weeks after the lastimmunization. In order to challenge the immunized mice with B. afzelii,the hair on the back of each mouse was removed with Veet® Cream (ReckittBenckiser, United Kingdom) and a small ventilated container was glued tothe skin with super glue (Pattex, Germany). Thereafter, one or two I.ricinus nymphs infected with B. afzelii, strain IS1, were applied permouse, allowed to attach and feed to depletion. The feeding status wasmonitored daily for each individual tick and only mice where at leastone fully-fed tick was collected were included in the final readout. Nodistinction was made between mice where one or two fully-fed ticks werecollected.

Six weeks after the tick application, blood was collected by orbitalbleeding and final sera were prepared and used for VISE ELISA analysisto determine infection status. The mice were then sacrificed by cervicaldislocation and one ear from each mouse was collected, DNA extracted andsubjected to nested PCR analysis to identify Borrelia in tissue.

Infection Readout

Only mice where the applied tick(s) fed to completion and could becollected were included in the final readout of the experiment. The micewere sacrificed 6 weeks after tick application and organs as well asfinal sera were collected. The final infection readout was based on twodifferent analyses (nested PCR targeting the 16S-23S intergenic spacerand VIsE (IR6) ELISA as described in detail below).

Nested PCR Targeting the 16S-23S Intergenic Spacer

One ear from each mouse was subjected to DNA extraction and purificationusing the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to themanufacturer's instructions, with the following modification. Each earwas digested over night at 60° C. in recombinant Proteinase K, PCR grade(Roche, 14-22 mg/mL). The DNA was eluted in 50 μL deionized sterilewater and stored at −20° C. until further analysis. As a negativecontrol, one empty purification column was included in each DNAextraction and purification and the eluate subjected to nested PCR. AllDNA extracts were screened for the presence of Borrelia DNA by a nestedPCR procedure, comprising 40 cycles of 94° C. for 30 s, 56° C. for 30 sand 72° C. for 60 s using the primers; Forward5′-GTATGTTTAGTGAGGGGGGTG-3′ (SEQ ID NO: 26) and Reverse5′-GGATCATAGCTCAGGTGGTTAG-3′ (SEQ ID NO: 27). From the reaction volumeof 10 μL, 1 μL was used as template for the nested PCR reaction. Thenested PCR step comprised 25 cycles of 94° C. for 30 s, 60° C. for 30 sand 72° C. for 60 s using the primers; Forward nested 5′AGGGGGGTGAAGTCGTAACAAG-3′ (SEQ ID NO: 28) and Reversed nested5′-GTCTGATAAACCTGAGGTCGGA-3′ (SEQ ID NO: 29). Of the final reactionvolume, 5 μL was separated on a 1% agarose gel containing ethidiumbromide and bands were visualized in UV-light.

In each PCR analysis, DNA purified from an in vitro grown culture of B.afzelii strain K78 was used as a positive control template. In addition,PBS was used instead of extracted DNA as negative control. Fivemicroliters of the final product was separated on a 1% agarose gelcontaining ethidium bromide and bands were visualized in UV-light.

ELISA with the Invariable Region 6 (IR6) of the Variable MajorProtein-Like Sequence E Protein (VIsE)

A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) (SEQ ID NO:30) derived from the sequence of B. garinii strain IP90 was used foranalysis (Liang F T, et al. (1999) J Immunol. 163:5566-73). Streptavidinpre-coated 96-well ELISA plates (Nunc, Denmark) were coated with 100μL/well (1 μg/mL) biotinylated peptide in PBS supplemented with 0.1%Tween 20 (PBS/0.1T). The plates were incubated overnight at 4° C. Aftercoating with the peptide, the plates were washed once with PBS/0.1T. Theplates were then blocked for one hour at room temperature (RT) with 100μL/well of PBS+2% BSA, before being washed again with PBS/0.1T.Reactivity of post-challenge sera to the peptide was tested at 1:200,1:400 and 1:800 dilutions in PBS+1% BSA. Plates were incubated for 90min at RT before being washed three times with PBS/0.1T. Each well thenreceived 50 μL of 1.3 μg/mL polyclonal rabbit anti-mouse IgG conjugatedto HRP (Dako, Denmark) in PBS+1% BSA. The plates were then incubated for1 h at RT. After three washes with PBS/0.1T, ABTS (50 μL/well) was addedas substrate (Sigma-Aldrich, USA) and color was allowed to develop for30 min. Absorbance was measured at 405 nm. All sera were tested induplicate; negative controls included PBS instead of sera, as well asplates not coated with the peptide. Sera from mice shown to be culturepositive for B. afzelii infection were used as positive controls.

Results Levels of Protection in the Tick Challenge Method

High levels of protection were observed for all five stabilized OspA B.afzelii fragments at both of the doses tested (30 μg and 5 μg, sec Table2). The high infection rates in the PBS control group indicate that theticks were infected with high frequency. Additionally, the positivecontrol, non-lipidated full-length OspA from B. afzelii strain K78, wasvery protective. Together these control groups indicate the highreliability of the experimental readout.

Protection results from experiments testing 30 μg doses (11 totalexperiments) and 5 μg doses (4 total experiments) are summarized inTable 2. The two methods employed to verify infection, namely VIsE ELISAand nested PCR, gave virtually identical results (data not shown),demonstrating the robustness of these readout methods for assessinginfection in the Tick Challenge Method.

Example 3. Assessment of the Protective Capacity of Lipidated His-TaggedMutant Serotype 2 OspA Fragment Monomers Against In Vivo BorreliaChallenge Via the Tick Challenge Method (ST2, B. afzelii) ExperimentalProcedures Cloning and Expression of Lipidated His-Tagged Mutant (SpaFragment Proteins

The serotype 2 mutant OspA fragments with cysteine bond types 1, 3 and 4(SEQ ID NOs: 141, 143 and 144, respectively) were modified by theaddition of a lipidation signal sequence derived from OspA (SEQ ID NO:14) and followed directly C-terminally by a CKQN peptide (SEQ ID NO:211) to provide an N-terminal cysteine for lipidation. All mutant OspAfragments were C-terminally histidine-tagged for purification purposes.Gene fragments were cloned into the pET28b(+) vector (Novagen), a vectorcontaining a Kanamycin resistance cassette as well as a T7 promoter. Thelipidated monomers were expressed in BL21 Star™ (DE3) cells (Invitrogen)and after induction by IPTG, the growth temperature of the cells waslowered from 37° C. to 25° C. to promote efficient post-translationalprocessing of the proteins. Cells were collected after 4 h bycentrifugation and the pellet was stored at −70° C. for up to 12 monthsprior to further processing.

Purification of Lipidated His-Tagged Wild-Type and Mutant OspA FragmentMonomer Proteins

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated His-tagged OspA fragment monomer polypeptides wereenriched in the lipid phase by phase separation, using Triton X-114 asdetergent. Subsequently, the diluted detergent phase (20 to 30 fold) wasapplied to a Ni-sepharose column (Ni Sepharose™ 6 Fast Flow; GEHealthcare) and the lipidated His-tagged OspA fragments were eluted byImidazole gradient (0-250 mM) elution. Pooled fractions were furtherpurified over a gel filtration column (Superdex 200, GE Healthcare)followed by a buffer exchange column (Sephadex G-25, GE Healthcare).Lipidated His-tagged OspA fragment peaks were pooled on the basis of theanalytical size exclusion column and reversed phase chromatography.After sterile filtration, the purified proteins were stored at −20° C.until formulation.

Immunization of Mice

Three lipidated mutant OspA proteins (Lip-S2D1-His, Lip-S2D3-His andLip-S2D4-His) were expressed and purified as described above. In vivoprotection studies were performed as described in Example 2 usingAl(OH)₃-adjuvant alone and non-lipidated full-length serotype 2 OspA asnegative and positive controls, respectively. All immunogens wereformulated with 0.15% Al(OH)₃. Mice were injected subcutaneously threetimes at two week intervals with formulations containing 3.0 μg. 1.0 μgor 0.3 μg antigen and challenged with B. afzelii-infected ticks (strainIS1) two weeks after the last immunization. Mice were sacrificed sixweeks following tick challenge and infection was assessed.

Results Levels of Protection in the Tick Challenge Method

All three lipidated mutant OspA fragments conferred very high levels ofprotection from B. afzelii challenge even at the lowest tested dose(Table 3). Infection rates in the Al(OH)₃-adjuvant alone immunized micewere high, indicating that the ticks were infected to a high frequency.The positive control antigen, full-length non-lipidated OspA from B.afzelii strain K78, was also very protective. Together, these controlgroups indicate the high reliability of the method of infection and thusgive high credibility to the results observed following immunizationwith the lipidated mutant OspA fragments.

Example 4. Assessment of the Protective Capacity of Mutant OspAHeterodimers of the Invention Against In Vivo Borrelia Challenge Via theNeedle or Tick Challenge Methods Experimental Procedures Cloning andExpression of Lipidated His-Tagged Mutant OspA Fragment Heterodimers

The mutant OspA fragment monomers from B. burgdorferi s.s. strain B31,B. afzelii strain K78, B. garinii strain PBr, B. bavariensis strain PBi,B. garinii strain PHEi and B. garinii strain DK29 were codon-optimizedfor E. coli expression by GenScript, USA. The hLFA-1-like epitope (aa164-174, SEQ ID NO: 17) of the OspA from B. burgdorferi s.s. strain B31was replaced by a non-hLFA-1-like sequence NFTLEGKVAND from B. afzeliistrain K78 (SEQ ID NO: 18). The lipidation signal sequence added to themutant OspA fragment heterodimers was derived from the E. coli majorouter membrane lipoprotein, Lpp, and was followed directly C-terminallyby a CSS peptide to provide an N-terminal cysteine for lipidation. Themutant OspA fragment heterodimers were generated by fusing differentmutant OspA fragment monomers as described above via a 21 amino acidlinker sequence, originating from two separate loop regions of theN-terminal half of OspA from B. burgdorferi s.s. strain B31 (“LN1”; as65-74 and as 42-53 with an amino acid exchange of D53S, SEQ ID NO: 184).The heterodimers were constructed with a His-tag for purificationpurposes. Gene fragments were cloned into the pET28b(+) vector(Novagen), a vector containing a Kanamycin resistance cassette as wellas a T7 promoter. The lipoproteins of the stabilized heterodimers wereexpressed in BL21 Star™ (DE3) cells (Invitrogen) and after induction byIPTG, the growth temperature of the cells was lowered from 37° C. to 25°C. to promote efficient post-translational processing of the proteins.Cells were collected after 4 h by centrifugation and the pellet wasstored at −70° C. for up to 12 months prior to further processing.

Purification of Lipidated His-Tagged Mutant OspA Fragment Heterodimers

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated His-tagged mutant OspA fragment heterodimers were enrichedin the lipid phase by phase separation, using Triton X-114 as detergent.Subsequently, the diluted detergent phase (20 to 30 fold) was applied toa Ni-sepharose column (Ni Sepharose™ 6 Fast Flow; GE Healthcare) and thelipidated His-tagged OspA heterodimers were eluted by Imidazole gradient(0-250 mM) elution. Pooled fractions were further purified over a gelfiltration column (Superdex 200, GE Healthcare) followed by a bufferexchange column (Sephadex G-25, GE Healthcare). The lipidated His-taggedmutant OspA heterodimer peaks were pooled on the basis of the analyticalsize exclusion column and reversed phase chromatography. After sterilefiltration, the purified heterodimers were stored at −20° C. untilformulation.

Cloning and Expression of Lipidated Non-His-Tagged Mutant OspA FragmentHeterodimers

The constructs made as described as above were used for the generationof constructs without a His-tag by the introduction of a stop cotton byPCR amplification. Gene fragments were cloned into the pET28b(+) vector(Merck Millipore), a vector containing a kanamycin resistance cassetteas well as a T7 promoter. The lipoproteins of the stabilizedheterodimers were expressed in BL21 Star™ (DE3) cells (Invitrogen) andafter induction by IPTG, the growth temperature of the cells was loweredfrom 37° C. to 25° C. to promote efficient post-translational processingof the proteins. Cells were collected after 4 h by centrifugation andthe pellet was stored at −70° C. for up to 12 months prior to furtherprocessing.

Purification of Lipidated Non-His-Tagged Mutant OspA FragmentHeterodimers

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated mutant OspA fragment heterodimers were enriched in thelipid phase by phase separation, using Triton X-114 as detergent.Subsequently, the diluted detergent phase was subjected to anionexchange chromatography operated in non-binding mode. The resultingflow-through was loaded on a hydroxyapatite column (Bio-Rad) and thelipidated proteins were eluted from the column by a linear saltgradient. The eluate was subjected to further purification over aDEAE-Sepharose column (GE Healthcare) in non-binding mode followed bygel filtration column (Superdex 200, GE Healthcare) for buffer exchange.The lipidated mutant OspA heterodimer peaks were pooled on the basis ofthe analytical size exclusion column and SDS-PAGE. After sterilefiltration, the purified heterodimers were stored at −20° C. untilformulation.

Immunization of Mice

Female C3H/HeN mice (Janvier, France) were used for all studies. Priorto each challenge, groups of ten 8-week-old mice were bled via thefacial vein and pre-immune sera were prepared and pooled. Threesubcutaneous (s.c.) immunizations of 100 μL each were administered attwo week intervals. Each dose contained the amount of immunogenindicated in Table 4 (dose), formulated with aluminium hydroxide(Al(OH)₃) at a final concentration of 0.15%. One week after the thirdimmunization, blood was collected from the facial vein and hyper-immunesera were prepared. In each experiment, one group immunized with Al(OH)₃alone was included as a negative control. All animal experiments wereconducted in accordance with Austrian law (BGB1 Nr. 501/1989) andapproved by “Magistratsabteilung 58”.

Tick Challenge of Immunized Mice and Collection of Sera and Tissues(Herein Referred to Also as “Tick Challenge Method”)

In order to challenge the immunized mice with B. afzelii, the hair ofthe back of each mouse was removed with Veet® Cream (Reckitt Benckiser)and a small ventilated container was glued to the skin with super glue(Pattex). Thereafter, one or two I. ricinus nymphs infected with B.afzelii, strain IS1, were applied per mouse, allowed to attach and feeduntil they were fully engorged and dropped off. The feeding status wasmonitored daily for each individual tick and only mice from which atleast one fully-fed tick was collected were included in the finalreadout.

Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

Two weeks after the last immunization, the mice were challenged s.c.with Borrelia diluted in 100 μL Borrelia growth medium (BSK II). Thechallenge doses were strain-dependent, the virulence of the individualstrains being assessed by challenge experiments for determination ofID₅₀. Doses employed for needle challenge experiments ranged from 20 to50 times the ID₅₀.

Sacrifice of Mice and Collection of Material

Four weeks after needle challenge with the indicated Borrelia App. orsix weeks after tick challenge with B. afzelii, mice were sacrificed bycervical dislocation. The blood was collected by orbital bleeding andfinal sera were prepared and used for VIsE ELISA to determine infectionstatus. In addition, one ear from each mouse was collected, and DNA wasextracted and subjected to quantitative PCR (qPCR) for identification ofBorrelia. The final infection readout was based on two differentanalyses (VIsE ELISA and qPCR targeting recA).

ELISA with the Invariable Region 6 (IR6) of VIsE

A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) derived fromthe sequence of B. garinii strain IP90 was used for the analysis (LiangF T, Alvarez A L, Gu Y, Nowling J M, Ramamoorthy R, Philipp M T. Animmunodominant conserved region within the variable domain of VISE, thevariable surface antigen of Borrelia burgdorferi. J Immunol. 1999;163:5566-73). Streptavidin pre-coated 96-well ELISA plates (Nunc), werecoated with 100 μL/well (1 μg/mL) peptide in PBS supplemented with 0.1%Tween (PBS/0.1T). The plates were incubated overnight at 4° C. Aftercoating with the peptide, the plates were washed once with PBS/0.1T. Theplates were then blocked for one hour at room temperature (RT) with 100μL/well of PBS+2% BSA, before being washed again with PBS/0.1T.Reactivity of post-challenge sera to the peptide was tested at 1:200,1:400 and 1:800 dilutions in PBS+1% BSA. Plates were incubated for 90min at RT before being washed three times with PBS/0.1T. Each well thenreceived 50 μL of 1.3 μg/mL polyclonal rabbit anti-mouse IgG conjugatedto HRP (Dako) in PBS+1% BSA. The plates were then incubated for 1 h atRT. After three washes with PBS/0.1T, ABTS (50 μL/well) was added assubstrate (Sigma-Aldrich) and color was allowed to develop for 30 min.Absorbance was measured at 405 nm. All sera were tested in duplicate.Negative controls included PBS instead of sera as well as plates notcoated with the peptide. Sent from mice shown to be culture positive forB. afzelii infection were used as positive controls.

qPCR Targeting recA

Oligonucleotide primers were designed for the recA gene in a manner thatthey could be used in qPCR for identification of all relevant Borreliaspecies causing Lyme borreliosis (forward: CATGCTCTTGATCCTGTTTA,reverse: CCCATTTCTCCATCTATCTC). The recA fragment was cloned from the B.burgdorferi s.s. strain N40 into pET28b(+), to be used as standard ineach reaction. The chromosomal DNA extracted from mouse cars was diluted1:8 in water in order to reduce matrix effects observed with undilutedDNA. A master mix consisting of 10 μL SSoAdvanced™ SYBR® Green Supermix,0.3 μL of each primer (10 μM), and 7.4 μL water was prepared for eachexperiment. Eighteen μL of master mix was mixed with 2 ILL of thediluted DNA extracted from either bladder or car in micro-titer platesand the DNA was amplified using a CFX96 real-time PCR detection system(Bio-Rad, USA). The DNA was denatured for 3 minutes at 95° C., followedby 50 cycles of 15 seconds at 95° C. and 30 seconds at 55° C. Afteramplification, the DNA was prepared for the melting curve analysis bydenaturation for 30 seconds at 95° C. followed by 2 minutes at 55° C.The melting curve analysis was performed by 5 seconds incubation at 55°C., with a 0.5° C. increase per cycle, and 5 seconds at 95° C. On eachplate, four no-template controls (NTC) were included as well as astandard curve in duplicate with template copy numbers ranging from 10to 10,000.

Results

Lipidated mutant OspA fragment heterodimers were tested for protectivecapacity in twelve separate experiments. Mice were challenged witheither B. burgdorferi s.s., strain N40, OspA serotype 1 (ST1, needlechallenge) or B. afzelii strain IS1, OspA serotype 2 (ST2, tickchallenge) in three experiments each or B. bavariensis, strain Sef, OspAserotype 4 (ST4, needle challenge), B. garinii, strain “A”, OspAserotype 5 (ST5, needle challenge) or B. garinii, strain “B”, OspAserotype 6 (ST6, needle challenge) in two experiments each. In allexperiments, a group of mice immunized with Al(OH)₃ adjuvant aloneserved as a negative control group. For challenge with ticks. 1-2 tickswere applied per mouse and only mice from which at least one tick feduntil fully engorged were included in the final readout. However, nodistinction was made between mice from which one or two fully-fed tickswere collected. The protection data from the twelve experiments aresummarized in Table 4.

The lipidated His-tagged OspA heterodimer (Lip-S1D1-S2D1-His) showedhighly statistically-significant protection (Fisher's exact test,two-tailed) in all six experiments against both OspA serotype 1 and OspAserotype 2 challenge as compared to the negative control group.Surprisingly, immunization with Lip-S4D1-S3D1-His and Lip-S5D1-S6D1-Hisalso conferred a high protective capacity against OspA serotype 2challenge (Experiments 4-6), indicating that there can be across-protective effect of immunization with other serotypes of themutant OspA fragments. Furthermore, immunization with Lip-S4D1-S3D1conferred statistically-significant protection against needle challengewith OspA serotype 4 Borrelia (Experiments 7 and 8). Finally,immunization with Lip-S5D1-S6D1-His conferred protection against needlechallenge with both OspA serotype 5 (Experiments 9 and 10) and OspAserotype 6 (Experiments 11 and 12). The infectious status of each mousewas determined using VIsE ELISA in combination with recA qPCR. A mousewas regarded as infected when at least one method gave a positiveresult.

In conclusion, immunization with mutant OspA fragment heterodimerpolypeptides of the invention confers protection against all Borreliaserotypes tested and also may provide cross-protection in some cases.

The lipidated His-tagged OspA heterodimer (Lip-S1D1-S2D1-His) showedhighly statistically-significant protection (Fisher's exact test,two-tailed) in all six experiments against both OspA serotype 1 and OspAserotype 2 challenge as compared to the negative control group.Surprisingly, immunization with Lip-S4D1-S3D1-His and Lip-S5D1-S6D1-Hisalso conferred a high protective capacity against OspA serotype 2challenge (Experiments 4-6), indicating that there can be across-protective effect of immunization with other serotypes of themutant OspA fragments. Furthermore, immunization with Lip-S4D1-S3D1conferred statistically-significant protection against needle challengewith OspA serotype 4 Borrelia (Experiments 7 and 8). Finally,immunization with Lip-S5D1-S6D1-His conferred protection against needlechallenge with both OspA serotype 5 (Experiments 9 and 10) and OspAserotype 6 (Experiments 11 and 12). The infectious status of each mousewas determined using VIsE ELISA in combination with recA qPCR. A mousewas regarded as infected when at least one method gave a positiveresult.

In conclusion, immunization with mutant OspA fragment heterodimerpolypeptides of the invention confers protection against all Borreliaserotypes tested and also may provide cross-protection in some cases.

Example 5. Assessment of the Protective Capacity of a 1:1:1 CombinationVaccine of the Mutant OspA Heterodimers of the Invention Against In VivoOspA Serotype 1 and Serotype 2 Borrelia Challenge Via the NeedleChallenge or Tick Challenge Methods Experimental Procedures Immunizationof Mice

Female C3H/HeN mice (Janvier, France) were used for all studies. Priorto each challenge, groups of ten 8-week-old mice were bled via thefacial vein and pre-immune sera were prepared and pooled. Three s.c.immunizations of 100 μL each were administered at two week intervals.Groups of mice were immunized with the combination vaccine consisting of1 μg each of Lip-S1 D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1. Threeother OspA-based antigens were included in the challenge experiments:Lip-OspA1-His (full-length serotype 1 OspA, lipidated and his-tagged),lipidated chimeric OspA ST1/ST2* and Lip-S1D1-S2D1 alone. The negative(placebo) control was Al(OH)₃-adjuvant alone. All antigens wereformulated in PBS with aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. *(Chimeric OspA ST1/ST2 (SEQ ID NO: 2121 is anOspA chimera consisting of the first 10 amino acids of the N-terminalportion of OspB (strain B31), amino acids 11-200 of serotype 1 OspA,fused with the last 201-255 amino acids from the C-terminal portion ofserotype 2 OspA and wherein the hLFA-1-like sequence of the serotype 1OspA (146-170) is replaced with the homologous sequence from a serotype2 OspA The serotype 2 OspA sequence is followed by two amino acids whichare added because of the cloning site (XhoI) ahead of the stop codon inthe vector.)

One week after the third immunization, blood was collected from thefacial vein and hyper-immune sera were prepared. All animal experimentswere conducted in accordance with Austrian law (BGB1 Nr. 501/19891 andapproved by “Magistratsabteilung 58”.

Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

Two weeks after the last immunization, the mice were challenged s.c.with Borrelia spirochetes diluted in 100 μL growth medium (BSKII). Thechallenge doses were strain-dependent, the virulence of the individualstrains were assessed by challenge experiments for determination ofID₅₀. Doses employed for needle challenge experiments ranged from 20 to50 times the ID 0. Four weeks after needle challenge, mice weresacrificed and blood and tissues were collected for readout methods todetermine the infection status.

Tick Challenge of Immunized Mice and Collection of Sera and Tissues(Herein Referred to Also as “Tick Challenge Method”)

In order to challenge the immunized mice with B. afzelii, the hair ofthe back of each mouse was removed with Veet® Cream (Reckitt Benckiser,United Kingdom) and a small ventilated container was glued to the skinwith super glue (Pattex, Germany). Thereafter, one or two I. ricinusnymphs infected with B. afzelii, strain IS1, were applied per mouse,allowed to attach and feed until they are fully engorged and drop off.The feeding status was monitored for each individual tick and only micewhere at least one fully-fed tick was collected were included in thefinal readout.

Results

Lipidated mutant OspA fragment heterodimers that were not His-taggedwere combined at a 1:1:1 ratio and tested for protective capacityagainst Borrelia challenge. Immunized mice were challenged with B.afzelii (ST2, strain IS1, tick challenge) or with B. burgdorferi s.s.(ST1, strain ZS7, needle challenge) in two experiments each. OtherOspA-based antigens included Lip-S1D1-S2D2 in all four experiments andLip-OspA1-His and lipidated chimeric OspA ST1/ST2 in Experiments 15 and16. A group of mice immunized with Al(OH)₃ adjuvant alone served as anegative control group in each experiment. For challenge with ticks, 1-2ticks were applied per mouse and only mice from which at least one tickfed until fully engorged were included in the final readout. However, nodistinction was made between mice from which one or two fully fed tickswere collected. The protection data from the four experiments aresummarized in Table 5.

The combination vaccine containing three lipidated mutant OspA fragmentheterodimers at a 1:1:1 ratio conferred statistically-significantprotection (Fisher's exact test, two-tailed) in all four challengeexperiments as compared to the negative control group. The infectiousstatus of each mouse was determined using VISE ELISA in combination withrecA qPCR. A mouse was regarded as infected when at least one methodgave a positive result.

Example 6 Binding of Antibodies from the Sera of Mice Immunized withMutant OspA Fragment Heterodimers to the Cell Surface of BorreliaExperimental Procedures Immunization of Mice

Female C3H/HeN mice were used for all studies. Prior to each challenge,groups of twenty 8-week-old mice were bled via the facial vein andpre-immune sera were prepared and pooled. Three s.c. immunizations of100 μL each were administered at two week intervals. Each dose contained1 μg of each of the respective proteins: Lip-S1D1-S2D1, Lip-S4D1-S3D1and Lip-S5D1-S6D1 (combination vaccine), or 1 μg lipidated full-lengthOspA protein (ST1-ST6 as indicated) or 1 μg OspA heterodimer alone(Lip-S1D1-S2D1, Lip-S4D1-S3D1 or Lip-S5D1-S6D1, as indicated) adjuvantedwith aluminium hydroxide at a final concentration of 0.15%. The negative(placebo) control was Al(OH)₁ adjuvant alone. One week after the thirdimmunization, blood was collected from the facial vein and hyper-immunesera were prepared. All animal experiments were conducted in accordancewith Austrian law (BGB1 Nr. 501/1989) and approved by“Magistratsabteilung 58”.

Flow Cytometry to Assess Binding to Borrelia

Spirochetes (1×10⁶) were mixed with an equal volume of 4%paraformaldehyde and incubated for 2 hours at room temperature in a96-well plate (Nunclon 96U, Nunc). The plate was centrifuged for 5minutes at 2,000 g and the supernatant was discarded. Cells were washedwith 150 μL HBSS with 2% BSA (HBSS-B), centrifuged as above and thesupernatant was discarded. Mouse sera were heat inactivated byincubating them at 56° C. for 35 minutes. Heat-inactivated sera werediluted in HBSS-B and sterile filtered by centrifuging 4,000 g for 3minutes using Costar spin-X centrifuge tube filters (0.22 μm, Corning,USA). Spirochetes were dissolved in 100 μL serum and incubated for 45minutes at room temperature. The plate was centrifuged for 15 minutes at2,000 g and the supernatant was discarded. The cells were washed oncewith 150 μL HBSS-B and then dissolved in 100 μL, HBSS-B.

One microliter secondary antibody (PE conjugated goat anti-mouse IgG,Beckman Coulter, USA) was added to the cells and incubated at roomtemperature for 45 minutes in the dark. Spirochetes were washed oncewith 150 μL HBSS-B and then dissolved in 200 μL HBSS containing 2.5 μMSYTO-17 DNA dye and incubated for 10 minutes at room temperature in thedark. The stained spirochetes were pelleted by centrifuging for 5minutes at 2000 g and subsequently dissolved in 200 μL HBSS. Labelledspirochetes were measured with a FC500 (Beckman Coulter) flow cytometer,gated for SYTO-17 positive events. Values obtained with sera from theplacebo-immunized group were subtracted from the values observed withsera from the heterodimer-immunized groups to control for non-specificbinding.

Results

Binding of antibodies from hyperimmune mouse sera was observed in thecase of different Borreliae expressing all six OspA serotypes,indicating that the antibodies generated in response to all of theantigens are functionally active and can bind native OspA in situ. Thefluorescence intensity was linear over a large range of serum dilutions.For most OspA serotypes, the observed fluorescence intensity withheterodimer-generated sera was comparable to the fluorescence intensityseen with sera generated with lipidated full-length OspA.

Example 7 Formulation Studies

Studies regarding the formulation of the combination vaccine of theinvention were carried out in order to optimize stability. Differenttypes of buffers and stabilizers were tested at various concentrationsin combination with aluminium hydroxide and antigen. An optimalformulation of 40 μg/mL each of three heterodimers (120 μg totalprotein), 10 mM sodium phosphate, 150 mM sodium chloride, 10 mML-Methionine, 5% Sucrose, 0.05% Tween 20 (polysorbate 20) and 0.15%(w/v) aluminium hydroxide at pH 6.7±0.2 was determined.#

SEQUENCES SEQ ID NO: 1S2D0-His: amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-typesequence, C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 2S2D1-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa182 and 269), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH SEQ ID NO: 3S2D2-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa182 and 272), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KGLEHHHHHH SEQ ID NO: 4S2D3-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa244 and 259), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT CVEIKTLDE LKNALKGLEHHHHHH SEQ ID NO: 5S2D4-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa141 and 241), C-terminal His tag (GLEHHHHHH) ELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQ C TITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 6S2D5-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa165 and 265), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALKGLEHHHHHH SEQ ID NO: 7S2D6-His: aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa185 and 272), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT C TLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KGLEHHHHHH SEQ ID NO: 8S2D7-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa199 and 223), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT CALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 9S2D8-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa243 and 262), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDT C TVQKYDSAGTNLEGTAVE C KTLDELKNALKGLEHHHHHH SEQ ID NO: 10S2D9-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa184 and 204), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEG C VTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 11S2D10-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa201 and 214), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVACNDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 12S2D11-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa246 and 259), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITV C KYDSAGTNLEGT CVEIKTLDE LKNALKGLEHHHHHH SEQ ID NO: 13S2D12-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa167 and 178), C-terminal His tag (GLEHHHHHH)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT C EVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 14 Borrelia OspA lipidation signalMKKYLLGIGLILALIA SEQ ID NO: 15 Borrelia OspB lipidation signalMRLLIGFALALALIG SEQ ID NO: 16 E. coli lpp lipidation signalMKATKLVLGAVILGSTLLAG SEQ ID NO: 17hLFA-1-like sequence from B. burgdorferi s.s. strain B31 GYVLEGTLTAESEQ ID NO: 18 Non-hLFA-1-like sequence from B. afzelii strain K78NFTLEGKVAND SEQ ID NO: 19 B. afzelii (strain K78; OspA serotype 2)MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKSEQ ID NO: 20 B. burgdorferi s.s. (strain B31, OspA serotype 1)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNINGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKSEQ ID NO: 21 B. garinii (strain PBr, OspA serotype 3)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALKSEQ ID NO: 22 B. bavariensis (strain PBi, OspA serotype 4)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALKSEQ ID NO: 23 B. garinii (strain PHei, OspA serotype 5)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALKSEQ ID NO: 24 B. garinii (strain DK29, OspA serotype 6)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNINGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALKSEQ ID NO: 25 B. garinii (strain T25, OspA serotype 7)MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVETKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALKSEQ ID NO: 26 Forward Primer GTATGTTTAGTGAGGGGGGTG SEQ ID NO: 27Reverse Primer GGATCATAGCTCAGGTGGTTAG SEQ ID NO: 28Forward Nested Primer AGGGGGGTGAAGTCGTAACAAG SEQ ID NO: 29Reversed Nested Primer GTCTGATAAACCTGAGGTCGGA SEQ ID NO: 3025-mer peptide MKKDDQIAAAMVLRGMAKDGQFALK SEQ ID NO: 31 Mouse cathelinRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE SEQ ID NO: 32 5′-(dldC)₁₃-3′dldC dldC dldC dldC dldC dldC dldG dldC dldC dldC dldC dldC dldCSEQ ID NO: 33 KLK peptide KLKLLLLLKLK SEQ ID NO: 34B. afzelii (strain K78, serotype 2), OspA aa 126-273FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 35B. afzelii (strain K78, serotype 2), OspA aa 131-273ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 36 peptide linker GGGGGGGG SEQ ID NO: 37peptide linker GGGGGGGGGGGG SEQ ID NO: 38 peptide linker GAGASEQ ID NO: 39 peptide linker GAGAGAGA SEQ ID NO: 40 peptide linkerGAGAGAGAGAGA SEQ ID NO: 41 peptide linker GGGSGGGS SEQ ID NO: 42peptide linker GGGSGGGSGGGS SEQ ID NO: 43S1D4-S2D4_aa: Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bond type4, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequenceNFTLEGKVANDFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 44Lip-S1D4-S2D4_nt: Coding sequence for fusion proteins of OspA serotypes 1 and 2 both withdisulfide bond type 4, E. coli lpp lipidation signal, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAA SEQ ID NO: 45Lip-S1D4-S2D4_His_aa: Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfidebond type 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag(GLEHHHHHH)LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLSEQ ID NO: 46Lip-S1D4-S2D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 1 and 2both with disulfide bond type 4, E. coli lpp lipidation signal, LN1 linker sequence, aa 164-174 ofOspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag(GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 47S1D1-S2D1_aa: Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfidebond type 1, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-likesequence NFTLEGKVANDFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 48Lip-S1D1-S2D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 1 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAA SEQ ID NO: 49Lip-S1D1-S2D1_His_aa: Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 withdisulfide bond type 1, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminalHis tag (GLEHHHHHH)LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHHSEQ ID NO: 50Lip-S1D1-S2D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 1 andOspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag(GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 51S3D4-S4D4_aa: Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfidebond type 4. LN1 linker sequenceFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNINGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 52Lip-S3D4-S4D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 3 and OspA serotype 4 with disulfide bond type 4, E. coli lpp lipidation signal, LN1 linkersequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGSEQ ID NO: 53Lip-S3D4-S4D4_His_aa: Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 withdisulfide bond type 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linkersequence, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHHSEQ ID NO: 54Lip-S3D4-S4D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 3 andOspA serotype 4 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 55S3D1-S4D1_aa: Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfide bond type1, LN1 linker sequenceFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 56Lip-S3D1-S4D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 3 and 4 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGSEQ ID NO: 57Lip-S3D1-S4D1_His_aa: Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfidebond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, N-terminallipidation, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVETKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHHSEQ ID NO: 58Lip-S3D1-S4D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 3 and 4both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 59S5D4-S6D4_aa: Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type 4,LN1 linker sequenceFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 60Lip-S5D4-S6D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins OspAserotypes 5 and 6 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGGGGGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAASEQ ID NO: 61Lip-S5D4-S6D4_His_aa: Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bondtype 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linker sequence, C-terminalHis tag (GLEHHHHHH)LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDETLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 62Lip-S5D4-S6D4_His_nt: Coding sequence for heterodimer fusion protein OspA serotypes 5 and 6both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 63S5D1-S6D1_aa: Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1, LN1linker sequenceFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 64Lip-S5D1-S6D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 6 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAASEQ ID NO: 65Lip-S5D1-S6D1_His_aa: Heterodimer fusion protein of OspA serotypes 6 both with disulfide bondtype 1, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linker sequence, C-terminalHis tag (GLEHHHHHH)LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDETLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 66Lip-S5D1-S6D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 6 bothwith disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids,LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 67S2D4-S1D4_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type4, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequenceNFTLEGKVANDFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 68Lip-S2D4-S1D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 2 and 1 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hl.FA-1-likesequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAA SEQ ID NO: 69Lip-S2D4-S1D4_His_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfidebond type 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 linker sequence, aa164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag(GLEHHHHHH)LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHHSEQ ID NO: 70Lip-S2D4-S1D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 2 and 1both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequenceNFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 71S2D1-S1D1_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type1, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1replaced by non-hLFA-1-like sequence NFTLEGKVANDFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIQNALK SEQ ID NO: 72Lip-S2D1-S1D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 2 and 1 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-likesequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAA SEQ ID NO: 73Lip-S2D1-S1D1_His_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfidebond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation, C-terminal His tag(GLEHHHHHH)LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHHSEQ ID NO: 74Lip-S2D1-S1D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 2 and 1both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequenceNFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 75S4D4-S3D4_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type4, N-terminal CSS for addition of lipids, LN1 linker sequenceFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 76Lip-S4D4-S3D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 4 and 3 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAASEQ ID NO: 77Lip-S4D4-S3D4_His_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfidebond type 4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNINGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHH SEQ ID NO: 78Lip-S4D4-S3D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 4 and 3both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 79S4D1-S3D1_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type1, LN1 linker sequenceFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 80Lip-S4D1-S3D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 4 and 3 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAASEQ ID NO: 81Lip-S4D1-S3D1_His_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfidebond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHH SEQ ID NO: 82Lip-S4D1-S3D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 4 and 3both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 83S6D4-S5D4_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type4, LN1 linker sequenceFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 84Lip-S6D4-S5D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 6 and 5 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAASEQ ID NO: 85Lip-S6D4-S5D4_His_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfidebond type 4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 86Lip-S6D4-S5D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 6 and 5both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 87S6D1-S5D1_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type1, LN1 linker sequenceFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 88Lip-S6D1-S5D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotypes 6 and 5 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAASEQ ID NO: 89Lip-S6D1-S5D1_His_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfidebond type 1, LN1 linker sequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHHSEQ ID NO: 90Lip-S6D1-S5D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotypes 6 and 5both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition oflipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 91S1D4-S2D1_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 and OspAserotype 2 with disulfide bond type 1, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVANDFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 92Lip-S1D4-S2D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAA SEQ ID NO: 93Lip-S1D4-S2D1_His_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHHSEQ ID NO: 94Lip-S1D4-S2D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 1 withdisulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 95S1D1-S2D4_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 and OspAserotype 2 with disulfide bond type 4, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVANDFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 96Lip-S1D1-S2D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAA SEQ ID NO: 97Lip-S1D1-S2D4_His_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1and OspA serotype 2 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHHSEQ ID NO: 98Lip-S1D1-S2D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 1 withdisulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 99S3D4-S4D1_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 and OspAserotype 4 with disulfide bond type 1, LN1 linker sequenceFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 100Lip-S3D4-S4D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGSEQ ID NO: 101Lip-S3D4-S4D1_His_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVETKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHHSEQ ID NO: 102Lip-S3D4-S4D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 3 withdisulfide bond type 4 and OspÅ serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 103S3D1-S4D4_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 and OspAserotype 4 with disulfide bond type 1, LN1 linker sequenceFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 104Lip-S3D1-S4D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGSEQ ID NO: 105Lip-S3D1-S4D4_His_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHHSEQ ID NO: 106Lip-S3D1-S4D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 3 withdisulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 107S5D4-S6D1_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 and OspAserotype 6 with disulfide bond type 1, LN1 linker sequenceFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 108Lip-S5D4-S6D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAASEQ ID NO: 109Lip-S5D4-S6D1_His_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4and OspA serotype 6 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 110Lip-S5D4-S6D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 5 withdisulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 111S5D1-S6D4_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 and OspAserotype 6 with disulfide bond type 4, LN1 linker sequenceFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 112Lip-S5D1-S6D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGcTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAASEQ ID NO: 113Lip-S5D1-S6D4_His_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1and OspA serotype 6 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 114Lip-S5D1-S6D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 5 withdisulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 115S2D4-S1D1_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 and OspAserotype 1 with disulfide bond type 1, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVANDFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 116Lip-S2D4-S1D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAA SEQ ID NO: 117Lip-S2D4-S1D1_His_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4and OspA serotype 1 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHHSEQ ID NO: 118Lip-S2D4-S1D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 2 withdisulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced bynon-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 119S2D1-S1D4_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 and OspAserotype 1 with disulfide bond type 4, LN1 linker sequence, aa 164-174 of OspA serotype 1 replacedby non-hLFA-1-like sequence NFTLEGKVANDFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 120Lip-S2D1-S1D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVANDATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAASEQ ID NO: 121Lip-S2D1-S1D4_His_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1and OspA serotype 1 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHHSEQ ID NO: 122Lip-S2D1-S1D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 2 withdisulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced bynon-hLFA-1-like sequence NFTLEGKVAND, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 123S4D4-S3D1_aa: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 and OspAserotype 3 with disulfide bond type 1, LN1 linker sequenceFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNINGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 124Lip-S4D4-S3D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAASEQ ID NO: 125Lip-S4D4-S3D1_His_aa: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4and OspA serotype 3 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal CSS for addition of lipids, N-terminal lipidation, C-terminal His tag(GLEHHHHHH)LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHHSEQ ID NO: 126Lip-S4D4-S3D1_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 4 withdisulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 127S4D1-S3D4_aa: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 and OspAserotype 3 with disulfide bond type 4, LN1 linker sequenceFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 128Lip-S4D1-S3D4_nt: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 andOspA serotype 3 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS foraddition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAASEQ ID NO: 129Lip-S4D1-S3D4_His_aa: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1and OspA serotype 3 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHHSEQ ID NO: 130Lip-S4D1-S3D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 4 withdisulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 131S6D4-S5D1_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 and OspAserotype 5 with disulfide bond type 1, LN1 linker sequenceFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 132Lip-S6D4-S5D1_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAASEQ ID NO: 133Lip-S6D4-S5D1_His_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4and OspA serotype 5 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 134Lip-S6D4-S5D1 His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 6 withdisulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 135S6D1-S5D4_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 and OspAserotype 5 with disulfide bond type 4, LN1 linker sequenceFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 136Lip-S6D1-S5D4_nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, E. coli lpplipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequenceATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAASEQ ID NO: 137Lip-S6D1-S5D4_His_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1and OspA serotype 5 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidation, C-terminal His tag (GLEHHHHHH)LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHHSEQ ID NO: 138Lip-S6D1-S5D4_His_nt: Coding sequence for heterodimer fusion protein of OspA serotype 6 withdisulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, E. coli lpp lipidation signal,N-terminal CSS for addition of lipids, LN1 linker sequence, C-terminal His tag (GLEHHHHHH)ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 140Lip-S2D0-His: amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2,wild-type sequence, N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 141Lip-S2D1-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1(aa 182 and 269), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDEL C NALKGLEHHHHHH SEQ ID NO: 142Lip-S2D2-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2(aa 182 and 272), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KGLEHHHHHH SEQ ID NO: 143Lip-S2D3-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3(aa 244 and 259), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT CVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 144Lip-S2D4-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4(aa 141 and 241), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRE C GTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQ C TITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 145Lip-S2D5-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5(aa 165 and 265), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALKGLEHHHHHH SEQ ID NO: 146Lip-S2D6-His: aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6(aa 185 and 272), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT C TLSKEAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KGLEHHHHHH SEQ ID NO: 147Lip-S2D7-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7(aa 199 and 223), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT C ALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 148Lip-S2D8-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8(aa 243 and 262), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDT C TVQKYDSAGTNLEGTAVE C KTLDELKNALKGLEHHHHHH SEQ ID NO: 149Lip-S2D9-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9(aa 184 and 204), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEG C VTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 150Lip-S2D10-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10(aa 201 and 214), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVA C NDTNTTQATKKTCAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 151Lip-S2D11-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11(aa 246 and 259), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITV C KYDSAGTNLEGT CVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 152Lip-S2D12-His: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12(aa 167 and 178), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT C EVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 153Lip-S2D0: amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-typesequence, N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 154Lip-S2D1: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa182 and 269), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDEL C NALK SEQ ID NO: 155Lip-S2D2: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa182 and 272). N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK C GTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 156Lip-S2D3: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa244 and 259), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT CVEIKTLDELKNALK SEQ ID NO: 157Lip-S2D4: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa141 and 241), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQ C TITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 158Lip-S2D5: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa165 and 265), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALK SEQ ID NO: 159Lip-S2D6: aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa185 and 272), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT C TLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 160Lip-S2D7: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa199 and 223), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT C ALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 161Lip-S2D8: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa243 and 262), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDT C TVQKYDSAGTNLEGTAVE C KTLDELKNALK SEQ ID NO: 162Lip-S2D9: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa184 and 204), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEG C VTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 163Lip-S2D10: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa201 and 214), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVA C NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 164Lip-S2D11: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa246 and 259), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITV C KYDSAGTNLEGT CVEIKTLDELKNALK SEQ ID NO: 165Lip-S2D12: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa167 and 178), N-terminal CKQN for addition of lipidsLipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT C EVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 166S2D0: amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-typesequenceELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSATNLEGTAVEIKTLDELKNALK SEQ ID NO: 167S2D1: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa 182and 269)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LC NALK SEQ ID NO: 168S2D2: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa 182and 272) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 169S2D3: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa 244and 259)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT CVEIKTLDE LKNALK SEQ ID NO: 170S2D4: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa 141and 241) ELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQ C TITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 171S2D5: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa 165and 265) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN CTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALK SEQ ID NO: 172S2D6: aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa 185and 272) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT CTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 173S2D7: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa 199and 223)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT CALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 174S2D8: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa 243and 262)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDT C TVQKYDSAGTNLEGTAVE C KTLDELKNALK SEQ ID NO: 175S2D9: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa 184and 204) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEG CVTLSKEIAKSGEVT VALND CNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKSEQ ID NO: 176S2D10: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa 201and 214)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT VAC NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 177S2D11: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa 246and 259)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTIT C KYDSAGTNLEGT C VEIKTLDELKNALK SEQ ID NO: 178S2D12: aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa 167and 178) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT CEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 179B. burgdorferi s.s. (strain B31, serotype 1), OspA_aa 126-273 with replaced hLFA-like sequence fromserotype 1 OspAFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 180B. garinii (strain PBr, serotype 3), OspA_aa 126-274FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 181B. bavariensis (strain PBi, serotype 4), OspA_aa 126-273FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 182B. garinii (strain PHei, serotype 5), OspA_aa 126-273FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 183B. garinii (strain DK29, serotype 6), OspA_aa 126-274FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 184LN1 peptide linker constructed from two separate loop regions of the N-terminal half of OspA from B.burgdorferi s.s. strain B31 (aa 65-74 and aa 42-53, amino acid exchange at position 53: D53S)GTSDKNNGSGSKEKNKDGKYS SEQ ID NO: 185Lip-S1D4-S2D4_aa: Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bondtype 4, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1replaced by non-hLFA-1-like sequence NFTLEGKVANDLipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 186Lip-S1D1-S2D1_aa: Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 withdisulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspAserotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidationLipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNINGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 187Lip-S3D4-S4D4_aa: Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfidebond type 4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 188Lip-S3D1-S4D1_aa: Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfide bondtype 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 189Lip-S5D4-S6D4_aa: Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 190Lip-S5D1-S6D1_aa: Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1,N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 191Lip-S2D4-S1D4_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bondtype 4, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidationLipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNINGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 192Lip-S2D1-S1D1_aa: Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bondtype 1, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidationLipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNINGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 193Lip-S4D4-S3D4_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bondtype 4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNINGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 194Lip-S4D1-S3D1_aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bondtype 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 195Lip-S6D4-S5D4_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bondtype 4, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDETLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 196Lip-S6D1-S5D1_aa: Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bondtype 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNIVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDETLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 197Lip-S1D4-S2D1_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 andOspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidationLipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 198Lip-S1D1-S2D4_aa: Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 andOspA serotype 2 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidationLipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 199Lip-S3D4-S4D1_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 andOspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 200Lip-S3D1-S4D4_aa: Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 andOspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 201Lip-S5D4-S6D1_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 andOspA serotype 6 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNINGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 202Lip-S5D1-S6D4_aa: Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 andOspA serotype 6 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDETLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNIVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 203Lip-S2D4-S1D1_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 andOspA serotype 1 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidationLipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 204Lip-S2D1-S1D4_aa: Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 andOspA serotype 1 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND,N-terminal lipidationLipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNINGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 205Lip-S4D4-S3D1_aa: Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 andOspA serotype 3 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPÅEIKDLAELCAALK SEQ ID NO: 206Lip-S4D1-S3D4_aa: Coding sequence for intermediate and final heterodimer fusion proteins of OspAserotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, N-terminal CSSfor addition of lipids, LN1 linker sequence, N-terminal lipidationLipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 207Lip-S6D4-S5D1_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 andOspA serotype 5 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 208Lip-S6D1-S5D4_aa: Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 andOspA serotype 5 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 linkersequence, N-terminal lipidationLipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 209B. afzelii (strain K78; OspA serotype 2) aa 17-273, lipidation signal sequence removed (aa 1-16:MKKYLLGIGLILALIA), C-terminal His tag (GLEHHHHHH)CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHHSEQ ID NO: 210B. burgdorferi (OspA serotype 1, strain ZS7) aa 17-273, lipidation signal sequence removed (aa 1-16:MKKYLLGIGLILALIA), C-terminal His tag (LEHHHHHH)CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALKLEHHHHHHSEQ ID NO: 211 Cysteine-containing peptide from OspA CKQN SEQ ID NO: 212Chimeric OspA Serotype1/Serotype2, N-terminal lipidationLipCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKGTSDKNNGSGVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITMADGTRLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSMNISKSGEVSVELNDTDSSAATKKTAAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKLE SEQ ID NO: 213amino acids of positions 126-274 of B. garinii strain T25, OspA serotype 7FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK SEQ ID NO: 214Forward oligonucleotide primer for the RecA gene of BorreliaCATGCTCTTGATCCTGTTTA SEQ ID NO: 215 Histidine tag GLEHHHHHHSEQ ID NO: 216Reverse oligonucleotide primer for the RecA gene of BorreliaCCCATTTCTCCATCTATCTC

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

1.-48. (canceled)
 49. A polypeptide comprising a mutant fragment of aBorrelia outer surface protein A (OspA), wherein said mutant OspAfragment comprises SEQ ID NO: 219, or a variant thereof, wherein saidvariant has at least 95% sequence identity to a wild-type serotype 2OspA fragment defined by amino acid residues 126-273 of SEQ ID NO: 19,and wherein said variant differs from said wild-type serotype 2 OspAfragment at least by the addition of at least one disulfide bond. 50.The polypeptide according to claim 49, wherein said polypeptidecomprises a heterodimer selected from the group consisting of Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S1D4-S2D1(SEQ ID NO: 197), and Lip-S2D1-S1D4 (SEQ ID NO: 204).
 51. Thepolypeptide according to claim 49, wherein said polypeptide consists ofa heterodimer selected from the group consisting of Lip-S1 D1-S2D1 (SEQID NO: 186), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S1D4-S2D1 (SEQ ID NO:197), and Lip-S2D1-S1D4 (SEQ ID NO: 204).
 52. The polypeptide accordingto claim 49, wherein said polypeptide is Lip-S1D1-S2D1 (SEQ ID NO: 186).53. A pharmaceutical composition comprising the polypeptide according toclaim
 49. 54. The pharmaceutical composition according to claim 53,further comprising a pharmaceutically acceptable carrier or excipient.55. The pharmaceutical composition according to claim 54, wherein saidexcipient is L-methionine and/or aluminium hydroxide.
 56. A method oftreating or preventing against a Borrelia infection in a subject in needthereof comprising administering to the subject the polypeptide of claim49.
 57. The method according to claim 56, wherein the Borrelia isselected from the group consisting of B. burgdorferi, B. garinii, B.afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B.lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi, and B.sinica.
 58. A method of treating or preventing against a Borreliainfection in a subject in need thereof comprising administering to thesubject the pharmaceutical composition of claim
 53. 59. The methodaccording to claim 58, wherein the Borrelia is selected from the groupconsisting of B. burgdorferi, B. garinii, B. afzelii, B. andersoni, B.bavariensis, B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii,B. japonica, B. tanukii, B. turdi, and B. sinica.
 60. A method forproducing the polypeptide of claim 49, comprising: a) introducing avector encoding the polypeptide into a host cell; b) growing the hostcell under conditions allowing for expression of said polypeptide; c)homogenizing said host cell; and d) subjecting the host cell homogenateto purification steps.
 61. The method according to claim 60, whereinsaid vector is pET28b(+).
 62. The method according to claim 60, whereinsaid host cell is Escherichia coli (E. coli).
 63. The method accordingto claim 62, wherein said E. coli is an E. coli BL21 cell.
 64. Themethod according to claim 60, wherein said purification steps compriseenriching the polypeptide in a lipid phase separation.
 65. The methodaccording to claim 60, wherein said purification steps comprisepurifying the polypeptide over a gel filtration column.
 66. The methodaccording to claim 60, wherein said purification steps compriseprocessing the polypeptide over a buffer exchange column.